Microfluidic device and method of use

ABSTRACT

The current invention relates to the device and method to separate biological entities from a sample fluid by a microfluidic device. The claimed methods separate biological entities by differentiating the sizes of the biological entities with ultrasound modes. The claimed methods further utilize a multi-staged design that removes smaller size entities at earlier and wider sections and concentrates larger entities at later and narrower sections of a microfluidic channel.

BACKGROUND

The current invention generally relates to methods and apparatus toseparate biological entities, including cells, bacteria and moleculesfrom human blood, body tissue, body fluid and other human relatedbiological samples. The disclosed methods and apparatus may also beutilized to separate biological entities from animal and plant samples.More particularly, the current invention relates to the methods andapparatus for achieving separation of biological entities using one ormore of a micro-fluidic separation device/chip (“UFL”), and one or moreof a magnetic separation device (“MAG”), individually or in combination.For description purpose, “cells” will be used predominantly hereafter asa typical representation of biological entities in general. However, itis understood that the methods and apparatus disclosed in this inventionmay be readily applied to other biological entities without limitation.

Separation of biological entities from a fluid base solution, forexample separating a specific type of white blood cell from human blood,typically involves a first step of identifying the target biologicalentities with specificity, and followed by a second step of physicalextraction of the identified target biological entities from the fluidbase solution. In human blood, different types of biological cells mayhave various types of surface antigens or surface receptors, which arealso referred to as surface markers in this invention. Certain surfacemarkers on a given type of cell may be unique to said type of cell andmay be used to identify said type of cell from blood sample withspecificity.

FIG. 1A through FIG. 1C show examples of identifying or labeling targetcells 1 using superparamagnetic labels 2 (“SPLs”) as in FIG. 1A, usingoptical fluorescent labels 3 (“OFLs”) as in FIG. 1B, and using both theSPLs 2 and OFLs 3 together as in FIG. 1C.

In FIG. 1A, cell 1 has surface markers 11. SPLs 2 are conjugated withsurface antibodies or ligands, also referred to as “probes” 21, whichspecifically bind to the surface markers 11 of cell 1. Large quantity ofSPLs 2 having probes 21 are put into the solution where cell 1 resides.After incubation processes 9, a plurality of SPLs 2 are bound to cell 1surface with probes 21 selectively bound to surface markers 11 withspecificity. Thus, cells 1 is magnetically identified or labeled by SPLs2, i.e. magnetically labeled cell 10. A magnetic field with sufficientfield gradient may be applied to cell 10 to produce a physical force onthe SPLs 2 attached to the cell 10 surface. With sufficient strength,the physical force working through the SPLs 2 on cell 10 may be used toseparate and physically remove cell 10 from its liquid solution.

In FIG. 1B, cell 1 has surface markers 12. OFLs 3 are conjugated withprobes 22, which specifically bind to the surface markers 12 of cell 1.Large quantity of OFLs 3 having probes 22 are put into the solutionwhere cell 1 resides. After incubation processes 9, a plurality of OFLs3 are bound to cell 1 surface with probes 22 selectively bound tosurface markers 12 with specificity. Thus, cells 1 is opticallyidentified or labeled by OFLs 3, i.e. optically labeled cell 20. Byusing an optical based cell separation system, cell 1 may be separatedfrom its liquid solution based on the optical signal that OFL 3 producesunder an excitation light. One type of such optical based cellseparation system is a flow cytometer, wherein a liquid solution isstreamed through a conduit within said flow cytometer as a continuousflow. At least one excitation light source produces a light spot uponsaid liquid flow through said conduit at a first optical wavelength. Inthe presence of OFL 3 in the light spot, OFL3 is excited by firstwavelength and radiates optical light at a second wavelength. When cell1 with bound OFLs 3 passes said light spot within said flow, OFLs 3bound to cell 1 radiate optical signal in second wavelength. Thestrength of said optical signal as well as duration while cell 1 passesthe light spot may be used to identify the presence of cell 1 by theflow cytometer, which then diverts cell 1 into a second liquid flow pathor mechanically removes cell 1 from the liquid flow, thus separatingcell 1 from the fluid base. In practice, OFL 3 bound to cell 1 may bevarious types of fluorescent dyes or quantum dots, producing excitedoptical light at multiple wavelengths. A plurality of excitation lightsources may also be used in the same flow cytometer system to produceexcitation light spots at different locations of the liquid flow withdifferent excitation light wavelength. Combination of variouswavelengths produced by OFL 3 on same cell 1 may be used to increasespecificity of separation of cell 1, especially when a combination ofvarious types of surface markers 12 is needed to specifically identify asub-category target cell 1 population from a major category of same typeof cells, for example CD4-T cells from other white blood cells.

In FIG. 1C, cell 1 has both surface markers 11 and 12. SPLs 2 conjugatedwith probes 21 and OFLs 3 conjugated with probes 22 are both bound tocell 1 surface after incubation processes 9 to form magnetically andoptically labeled cell 30. Cell 30 allows for separation of cell 30 witha combination of a magnetic separation and an optical based cellseparation system. The magnetic separation through SPLs 2 may provide afast first stage separation of cell category including cell 30, whilethe optical separation through OFLs 3 may provide a second stageseparation of cell 30 after magnetic separation with more specificity.Alternatively, cell 30 may be separated via OFLs 3 in a first stage andvia SPLs 2 in a second stage. In either case, SPLs 2 and OFLs 3 togethermay help increase speed, efficiency and specificity in separation ofcell 1 compared with FIG. 1A and FIG. 1B.

FIG. 2A shows an example of conventional magnetic separation through SPL2. In a container 5, liquid solution 6 contains cells 10 of FIG. 1A orcells 30 of FIG. 1C that are bound with a plurality of SPLs 2 on cellsurface. Magnet 4, preferably a permanent magnet, is positioned inproximity to wall of container 5. Magnet 4 has a magnetizationrepresented by arrow 41 indicating a north pole (“N”) and a south pole(“S”) on top and bottom surfaces of the magnet 4. Magnetic fieldproduced by the magnetization 41 in the solution 6 is higher at thecontainer 5 wall directly opposing the N surface of the magnet 4, andlower at locations within solution 6 further away from the magnet 4,thus creating a magnetic field gradient pointing towards the magnet 4within the solution 6. SPLs 2 bound to cells 10/30 aresuperparamagnetic, which means that SPLs 2 are effectively non-magneticin the absence of magnetic field, but will gain magnetic moment in thepresence of the magnetic field produced by the magnet 4. With themagnetic moment of SPLs 2 and the magnetic field gradient from magnet 4,cells 10/30 will be pulled by the force produced by the magnetic fieldfrom magnet 4 towards magnet 4. After sufficient time 7, cells 10/30 maybe depleted from solution 6 and form conglomerate at inside surface ofthe container 5 wall opposing magnet 4. In conventional practice,solution 6 may be removed from container 5, while maintaining magnet 4position relative to container 5 thus cells 10/30 are retained asconglomerate against container 5 inside surface. Afterwards, magnet 4may be removed from container 5. With absence of magnetic field,conglomerate of cells 10/30, together with any non-bound free SPLs 2 inthe conglomerate, should self-demagnetize over an extensive period oftime to become non-magnetic and cells 10/30 may be removed fromcontainer 5 as individual cells 10/30.

Conventional method as shown in FIG. 2A has limitations in actualapplications. For the SPL 2 to be superparamagnetic, the size of thefundamental superparamagnetic particles (“SPN”) contained in SPL 2, forexample iron oxide particles, should be in the range of 10 nm(nanometer) to 30 nm. A smaller particle size makes the particles moreeffectively superparamagnetic but harder to gain magnetic moment in thepresence of magnetic field, while a larger particle size makes theparticles more difficult to become non-magnetic when magnetic field isremoved. SPL 2 is typically composed of SPNs dispersed in a non-magneticmatrix. For example, certain SPL 2 is a solid sphere formed by SPNsevenly mixed within a polymer base, typically has a size larger than 1μm. In another case, SPL 2 is a solid bead formed by SPNs mixed withinan oxide or nitride base, for example iron oxide nanoparticles mixed insilicon oxide base, which can have a size of a few hundred nanometers ortens of nanometers. For the cells 10/30 of FIG. 2A to be suitable foradditional cellular processes, including cell culture and cell analysis,SPL 2 size is desired to be smaller than the cell itself, which isusually a few micrometers. Thus, SPL 2 with sub-micrometer size (<1 μm)is desired. SPL 2 size less than 500 nm is more preferred. SPL 2 sizeless than 200 nm is most preferred. However, when SPL 2 average size issmaller, variation of SPL 2 size becomes larger statistically. FIG. 2Bshows an example plot of single SPL 2 magnetic moment in the presence ofan applied magnetic field. Solid curve 22 indicates SPL 2 having apopulation nominal size, or average size, where SPL 2 magnetic momentincreases with higher magnetic field. With magnetic field strengthincreasing from 0 to Hs, nominal size SPL magnetic moment increases withfield strength in a linear trend at beginning, until reaching asaturation region where magnetic moment plateaus to Ms, which isdetermined by the saturation moment of the SPN material within the SPL2. For SPL 2 with a smaller size than nominal size, curve 23 indicatesthat at the same magnetic field strength, smaller size SPL 2 gains alower moment, and thus experiences a lower magnetic force, and requiresa higher field to reach saturation magnetic moment Ms. For a larger sizeSPL 2 than nominal size, curve 24 indicates larger size SPL 2 is easierto saturate to Ms with a lower field and gains a higher moment at thesame magnetic field strength.

Now referring back to FIG. 2A, for SPL 2 with sub-micrometer size thatis suitable for cell separation and cellular processes, conventionalmethod of FIG. 2A has the limitation of not being able to produce highmagnetic field strength and strong magnetic field gradient in solution 6at locations further away from the container 5 wall opposing magnet 4 Nsurface. Therefore, smaller size SPL 2 of curve 23 of FIG. 2B at fartherend of the container 5 from magnet 4 may be difficult to magnetize bymagnetic field and experiences smaller force to move the cells 10/30towards magnet 4. To reach complete depletion of cells 10/30 in solution6 within container 5, it may require a significant amount of time.Meanwhile, volume of container 5 is limited also due to magnetic fieldstrength from magnet 4, which may not be sufficient to magnetize thesmaller SPL 2 of curve 23 of FIG. 2B at large container 5 sizes. Besidesthe overall process being slow, another drawback of conventional methodof FIG. 2A is that the operation as described in FIG. 2A typicallyinvolves air exposure of cell 10/30 conglomerate during the steps ofsolution removal and later removal of cells 10/30 from container 5. Suchair exposure poses challenge in achieving sterile separation of cells10/30 for clinical purpose, as well as risk of cell 10/30 damage ordeath that negatively affects further cellular processes of cells 10/30.

FIG. 3A shows another example of magnetic separation of cells 10/30 withSPL 2 in prior art. In FIG. 3A, solution 6 containing cells 10/30 ispassed through a column 31 that is filled with ferromagnetic orferrimagnetic spheres 36. By applying a magnetic field across the columnwith magnets 32 and 33, where dashed lines 34 indicates applied magneticfield direction, spheres 36 may be magnetized by the field and producelocalized magnetic field in gaps between neighboring spheres 36. Suchlocal field and field gradient between spheres 36 gaps may be strong,due to the small dimensions of the gaps, to effectively magnetize SPL 2of all sizes when SPL 2 in solution 6 passes through the gaps betweenthe spheres 36 during a downward flow of solution 6 as indicated byarrow 35, where SPL 2 may be attracted to various spheres 36 surface andseparated from the solution 6. Prior art of FIG. 3A may effectivelyavoid the air exposure issue of FIG. 2A, and may have a higherseparation speed of cells 10/30 than FIG. 2A during the flow 35.However, an intrinsic issue of FIG. 3A method is that with the spheres36 being ferromagnetic or ferrimagnetic and is much larger in size thancells 10/30, magnetic domains in spheres 36 will exist even afterremoval of magnets 32 and 33 from the column 31. Such magnetic domains,and domain walls between the domains, will inevitably produce localmagnetic fields around the surfaces of the spheres 36, which will keepthe SPLs 2 on cells 10/30 magnetized and strongly attract the cells10/30 when magnets 32 and 33 are removed. Therefore, the cells 10/30 areinherently more difficult to be removed from the column 31 in FIG. 3Athan FIG. 2A. Cell 10/30 loss due to incomplete removal from column 31after separation is inherently high. In certain prior art method, apressurized high speed buffer flow may be used to force wash the cells10/30 from the spheres in column 36. However, such forced flow willinevitably cause mechanical damage to the cells and will still leavebehind significant percentage of cells 10/30 in column 31 due to thestrong domain wall field of spheres 36. Besides cell 10/30 loss, anotherintrinsic issue of FIG. 3A method is introducing spheres 36 as foreignmaterials in the flow of solution 6, which is not desirable for sterileprocess needed for clinical applications.

FIG. 3B shows another prior art similar to method of FIG. 3A, exceptmesh 37 made of ferromagnetic or ferrimagnetic wires are introduced inthe column 31 instead of spheres or blocks 36. When magnetic field 34 isapplied by the magnets 32 and 33, wires of mesh 37 are magnetized andadjacent wires of mesh 37 produce local magnetic field around the wires.Clearances between wires of the mesh allow fluid 6 to flow in direction35 within the column. When cells 10/30 are in proximity to wires of mesh37, cells 10/30 may be attracted to the wire surface due to the localmagnetic field and field gradient produced by the wires of the mesh 37.Compared to FIG. 3A prior art, FIG. 3B may adjust size of wires and sizeof clearance of mesh 37 to tradeoff between cell 10/30 separation speedand cell loss in column. However, in practice, due to the gap betweenspheres 36 is much smaller than clearance size in mesh 37, cell 10/30separation speed in FIG. 3B is slower than FIG. 3A, while FIG. 3B stillhas the same cell loss issue of FIG. 3A, where domains in the wires ofmesh 37 maintains SPL 2 magnetic moment after magnets 32 and 33 areremoved and cells 10/30 are attracted to the wires by the domain anddomain wall. Cell 10/30 loss due to the magnetic domains in wires ofmesh 37 also exists in FIG. 3B. Additionally, FIG. 3B is same as FIG. 3Ain introducing mesh 37 as foreign materials in the flow of solution 6,which is not desirable for sterile process.

FIG. 3C shows another prior art, where magnets 32 and 33 are eachattached with a soft magnetic flux guide 38 with an apex. The fluxguides 38 produce localized magnetic field between the apexes of theguides 38 with high field strength and high gradient close to theapexes. FIG. 3C shows the cross-sectional view of the conduit 39, whichis intrinsically a circular tubing. The solution 6 containing cells10/30 flows along the tubing 39 length in the direction perpendicular tothe cross-section view. Tubing 39 is positioned on one side of the gapof the apexes. Magnetic field lines 34 exhibit a higher density closerto the gap, indicating both higher magnetic field strength and highermagnetic field gradient towards the gap. Magnetic field 34 produceseffective force on cells 10/30 in solution 6 and pulls the cells 10/30from solution 6 towards the tubing 39 inside wall that is closest to theapexes of the guides 38. Prior art of FIG. 3C when compared to prior artof FIG. 3A and FIG. 3B has the advantages of: (1) not introducingforeign material in the flow path; (2) when magnets 32 and 33 areremoved from tubing together with guides 38, there is no ferromagneticor ferrimagnetic sphere 36 or mesh 37 in the tubing, thus avoiding thedomain structures related loss of cells 10/30.

However, prior art of FIG. 3C also has intrinsic deficiencies. Firstdeficiency is the flow speed of solution 6, or flow rate, in the tubing39 is limited by the prior art design of FIG. 3C. The separation speedof cells 10/30 in the prior art of FIG. 3C is not sufficient for manyapplications. Circular tubing conduit 39 as shown in FIG. 3C experienceshigh field and high field gradient at lower end of tubing 39, wherecells 10/30 closer to the lower end of tubing 39 may experience a highforce that pulls them to move towards the tubing 39 lower wall innersurface much faster. However, for the cells 10/30 closer to the top endof the tubing 39, due to the narrow wedge gap and position of the tubing39 being on one side of the gap, magnetic field and gradient issignificantly lower than the lower end. Thus cells 10/30 closer to thetop end of the tubing 39 experience a much smaller force and move tolower end of tubing 39 at a much slower speed. For a limited length ofthe tubing 39 in the perpendicular to cross-sectional view direction,all cells 10/30 within the fluid 6 flowing through the tubing 39 need tobe separated from solution 6 to form a conglomerate on the insidesurface of the tubing close to the apexes before solution 6 exits thetubing 39. Due to slower speed of cells 10/30 moving from top of thetubing 39, flow rate of solution 6 needs to be slow such that it canallow enough time for all the cells 10/30 near top of tubing 39 to beattracted into the conglomerate. If solution 6 flows through the tubing39 at higher speeds, it will cause incomplete separation of cells 10/30from solution. Such limitation on flow rate due to the circular designof tubing 39, where tubing top end being further away from high fieldand high gradient apexes, cannot be cured by a smaller size tubing 39. Asmaller cross-sectional size circular tubing 39 will bring the top endof the tubing 39 closer to the wedge gap. However, due to the smallercross-section size, volume of solution 6 flowing through the tubing 39in a unit time frame, i.e. flow rate of solution 6, will reduce whenflow speed of solution 6 is maintained. To maintain the same flow rateas in a larger tubing 39, solution 6 flow speed needs to increase, whichthen gives less time for cells 10/30 at top end of smaller size tubing39 to move to the conglomerate site, and offsets the effect of smallsize tubing 39.

A second deficiency of FIG. 3C prior art is the inability to dissociateindividual cells 10/30 from conglomerate of cells 10/30 and non-boundfree SPL 2, as the conglomerate will not self-demagnetize with easeafter magnets 32 and 33, together will guides 38, are removed fromtubing 39 in actual applications. Demagnetization of SPL 2 relies on theSPNs within SPL 2 being effectively nanoparticles. However, as theconglomerate forms an effectively larger body of superparamagneticmaterial, the SPNs within SPL 2 experiences magneto-static field from alarge number of closely packed SPNs from neighboring SPLs 2 in theconglomerate, which reduces the super-paramagnetic nature of the SPNs.In one case, the SPL 2 of cells 10/30 within conglomerate requiresextensive time to self-demagnetize, which is not practical for manyapplications. In another case, the conglomerate won't self-demagnetizedue to the SPN being more ferromagnetic in conglomerate form, which isundesirable. High pressure flushing as utilized in FIG. 3A is noteffective in FIG. 3C, as majority of the circular tubing 39 inner areais occupied by empty space, while conglomerate is compacted on the lowerend of tubing 39. Such flush will mainly flow through the top section ofthe tubing 39 without producing enough friction force on theconglomerate of cells 10/30 to remove the cells 10/30 from the tubing 39lower wall. As prior art does not provide an effective method todissociate conglomerate and remove cells 10/30 from tubing 39, suchdeficiency of FIG. 3C prior art is limiting its application.

Prior art is limited either in causing cell loss and introducing foreignmaterials in the flow path, or limited in the flow rate of solution 6and the ability to extract separated cells from conglomerate with aneffective dissociation method.

It is desired to have a method and an apparatus that can achieve highflow rate magnetic separation of cells 10/30 without introducing foreignmaterial in the flow path of the biological solution, and are able todissociate cells 10/30 from conglomerate in a practically short timewithout damaging the cells.

SUMMARY OF THE INVENTION

This invention describes novel methods and novel magnetic separationdevices (“MAG”) that are able to: (1) separate biological entities boundwith SPLs from biological solution with high flow rate, without exposureof biological entities to air, and without introducing foreign materialin the flow path of the biological solution carrying the biologicalentities; (2) dissociate the biological entities from the magneticallyseparated conglomerate.

This invention further describes novel methods and novel micro-fluidicseparation devices (“UFL”) that separate biological entities frombiological solutions based on the size of the biological entities.

This invention further describes novel methods and novel apparatus usingMAG and UFL individually and in combination to separate biologicalentities from biological solutions for various separation applications.

This invention further describes methods of pre-symptom early tumordetection and methods of using MAG and UFL during the process of tumordetection.

The methods, components and apparatus as disclosed by this invention maybe utilized to separate biological entities, including cells, bacteriaand molecules, from human blood, human body tissue, human bones, humanbody fluid, human hairs, other human related biological samples, as wellas biological entities from animal and plant samples alike withoutlimitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates superparamagnetic labels (SPLs) binding to a cell.

FIG. 1B illustrates optical fluorescent labels (OFLs) binding to a cell.

FIG. 1C illustrates SPLs and OFLs binding to a cell.

FIG. 2A illustrates cells bound with SPLs being separated by a magnet.

FIG. 2B is a plot of SPL magnetization vs magnetic field strength fordifferent SPL sizes.

FIG. 3A is a cross-sectional view of a prior art magnetic cellseparator.

FIG. 3B is a cross-sectional view of a prior art magnetic cellseparator.

FIG. 3C is a cross-sectional view of a prior art magnetic cellseparator.

FIG. 4 is a cross-sectional view of the first embodiment of a magneticseparation device (“MAG”) with a “C” shape rigid channel.

FIG. 5 is a cross-sectional view of the first embodiment of MAG having a“C” shape rigid channel in separation position.

FIG. 6 is a cross-sectional view of the first embodiment of MAG having a“C” shape rigid channel in separation position with cells beingseparated.

FIG. 7 is a side view of FIG. 6 .

FIG. 8A is a cross-sectional view of the second embodiment of MAG.

FIG. 8B is a cross-sectional view of the third embodiment of MAG.

FIG. 9 a cross-sectional view of the first embodiment of MAG with aflexible channel.

FIG. 10 illustrates the first embodiment of MAG having a flexiblechannel in separation position with cells being separated.

FIG. 11 illustrates the first embodiment of MAG having a flexiblechannel in lifted position after cell separation.

FIG. 12 illustrates cross-sectional view of the fourth embodiment of MAGhaving a “D” shape rigid channel in separation position.

FIG. 13 illustrates cross-sectional view of the fourth embodiment of MAGwith a flexible channel.

FIG. 14 illustrates cross-sectional view of the fourth embodiment of MAGhaving a flexible channel in separation position with cells beingseparated.

FIG. 15A illustrates cross-sectional view of the fifth embodiment ofMAG.

FIG. 15B illustrates cross-sectional view of the sixth embodiment ofMAG.

FIG. 15C illustrates cross-sectional view of the seventh embodiment ofMAG.

FIG. 16 illustrates cross-sectional view of a pair of third embodimentof MAGs with a pair of flexible channels on a single channel holder.

FIG. 17 illustrates cross-sectional view of a pair of third embodimentof MAGs with a pair of flexible channels on a single channel holder inseparation position.

FIG. 18 illustrates cross-sectional view of four of fifth embodiment ofMAGs with four flexible channels on a single channel holder.

FIG. 19 illustrates cross-sectional view of four of fifth embodiment ofMAGs with four flexible channels on a single channel holder inseparation position.

FIG. 20A illustrates cross-sectional view of the eighth embodiment ofMAG with a rotated “D” shape rigid channel in separation position.

FIG. 20B illustrates cross-sectional view of the eighth embodiment ofMAG with a flexible channel.

FIG. 20C illustrates cross-sectional view of the eighth embodiment ofMAG having a flexible channel in separation position with cells beingseparated.

FIG. 21A illustrates cross-sectional view of the ninth embodiment of MAGhaving a “V” shape rigid channel in separation position.

FIG. 21B illustrates cross-sectional view of the ninth embodiment of MAGwith a flexible channel.

FIG. 21C illustrates cross-sectional view of the ninth embodiment of MAGhaving a flexible channel in separation position with cells beingseparated.

FIG. 22A illustrates the third embodiment of MAG having a flexiblechannel in separation position with cells being separated, and ademagnetization (“DMAG”) magnet positioned over and away from MAG.

FIG. 22B illustrates the flexible channel of FIG. 22A departing MAG andmoving into position where flexible channel holder is in close proximityto, or contacts, the DMAG magnet.

FIG. 22C illustrates the cells in the flexible channel of FIG. 22B beingdissociated from conglomerate by the DMAG magnet.

FIG. 22D illustrates the flexible channel of FIG. 22C moving into a lowmagnetic field position between MAG and DMAG magnet.

FIG. 23A illustrates mechanical vibration is applied to a flexiblechannel holder by a motor after cells are magnetically separated insidethe flexible channel.

FIG. 23B illustrates ultrasound vibration is applied to a flexiblechannel holder by a piezoelectric transducer (“PZT”) after cells aremagnetically separated inside the flexible channel.

FIG. 23C illustrates mechanical vibration being applied to a flexiblechannel by a motor after cells are magnetically separated inside theflexible channel.

FIG. 23D illustrates ultrasound vibration being applied to a flexiblechannel by a PZT after cells are magnetically separated inside theflexible channel.

FIG. 23E is a side view of the flexible channel of FIG. 22D.

FIG. 24A illustrates the third embodiment of MAG having a flexiblechannel holder in close proximity to, or in contact with, a DMAG magnetafter cells are magnetically separated by MAG, where DMAG magnet ispositioned on the side and away from MAG.

FIG. 24B illustrates the flexible channel of FIG. 24A rotating into alow magnetic field position between MAG and DMAG magnet.

FIG. 25A illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is a permanent magnet.

FIG. 25B illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is a permanent magnet attached with a softmagnetic pole.

FIG. 25C illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is a permanent magnet attached with a pairof soft magnetic poles.

FIG. 25D illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is an electro-magnet.

FIG. 25E illustrates flexible channel holder at demagnetizationposition, where mechanical vibration is applied to DMAG magnet by amotor.

FIG. 25F illustrates flexible channel holder at demagnetizationposition, where ultrasound vibration is applied to DMAG magnet by a PZT.

FIG. 26A illustrates the third embodiment of MAG having a flexiblechannel in separation position with cells being separated.

FIG. 26B illustrates the flexible channel of FIG. 26A departing from MAGand rotating.

FIG. 26C illustrates the conglomerate of separated cells in the flexiblechannel of FIG. 26B being rotated to top end of the flexible channel.

FIG. 26D illustrates the flexible channel of FIG. 26C moving todemagnetization position.

FIG. 27A illustrates the third embodiment of MAG having a flexiblechannel in separation position with cells being separated.

FIG. 27B illustrates the flexible channel and its holder of FIG. 27Adeparting from MAG.

FIG. 27C illustrates mechanical vibration being applied to the channelholder by a motor.

FIG. 27D illustrates ultrasound vibration being applied to the channelholder by a PZT.

FIG. 28A illustrates a side view of a flexible channel where theflexible channel is mechanically stretched.

FIG. 28B illustrates cells being dissociated from conglomerate afterremoval of the external force of FIG. 28A.

FIG. 29A illustrates a side view of a flexible channel where theflexible channel is mechanically compressed.

FIG. 29B illustrates cells being dissociated from conglomerate afterremoval of the external force of FIG. 29A.

FIG. 30A illustrates a side view of a flexible channel where theflexible channel is mechanically twisted.

FIG. 30B illustrates cells being dissociated from conglomerate afterremoval of the external force of FIG. 30A.

FIG. 31 is a schematic diagram illustrating methods to use MAG tomagnetically separate biological entities from fluid solution.

FIG. 32 illustrates a method to align a flexible channel MAG wedge of aMAG device.

FIG. 33A illustrates a flexible channel attached to the output port of aperistaltic pump, where a flow limiter is attached to the flexiblechannel to reduce the flow rate pulsation.

FIG. 33B illustrates a top-down view of the inner structure of a firsttype flow limiter.

FIG. 33C illustrates a side view of a second type flow limiter.

FIG. 34A illustrates FIG. 33A flow limiter being disengaged from theflexible channel.

FIG. 34B is a plot illustrating fluid flow rate with large pulsation.

FIG. 35A illustrates FIG. 33A flow limiter being engaged upon theflexible channel.

FIG. 35B is a plot illustrating fluid flow rate with reduced pulsation.

FIG. 36A illustrates FIG. 33A flow limiter causing pressure built up atthe fluid incoming end of the flow limiter.

FIG. 36B illustrates FIG. 36A flow limiter being disengaged and causinghigh speed fluid pulse that pushes the dissociated cells of FIG. 36A outof the channel.

FIG. 37 is a plot illustrating fluid flow rate pulse created by theprocess of FIG. 36A to FIG. 36B transition where flow limiter isdisengaged.

FIG. 38A is a top-down view of a micro-fluidic chip (“UFL”).

FIG. 38B is a cross-sectional view of a portion of the FIG. 38A UFLincluding entity fluid inlet, buffer fluid inlet, and part of the UFL.

FIG. 38C is a schematic diagram illustrating a single fluidic pressurenode created between two side walls of the UFL of FIG. 38A by ultrasoundvibration generated by a PZT.

FIG. 38D is a schematic diagram illustrating the fluid acoustic wave ofFIG. 38C causing larger size entities to move around center of the UFL.

FIG. 39 is a schematic diagram illustrating methods to use UFL toseparate biological entities of different sizes.

FIG. 40A is a cross-sectional view of a portion of first embodiment UFLwhich includes a uniformly formed soft magnetic layer.

FIG. 40B is a schematic diagram illustrating the fluid acoustic wave andentities entity separation in UFL of FIG. 40A in the presence of amagnetic field.

FIG. 40C is a schematic diagram illustrating a protection layerconformably deposited around the UFL surface before attaching cap.

FIG. 41A is a top-down view of a second embodiment UFL including a widechannel and a narrow channel in sequential arrangement.

FIG. 41B is a cross-sectional view of second embodiment UFL across widechannel.

FIG. 41C is a cross-sectional view of second embodiment UFL acrossnarrow channel.

FIG. 42A is a top-down view of a third embodiment UFL including a widechannel and a narrow channel, and side channels from wide channel tonarrow channel transition section.

FIG. 42B is a cross-sectional view of third embodiment UFL across widechannel.

FIG. 42C is a cross-sectional view of third embodiment UFL across narrowchannel and side channels.

FIG. 43 is a top-down view of a fourth embodiment UFL having three-stagechannel width reduction along channel flow direction, and side channelsfrom transition sections.

FIG. 44A illustrates first type sample processing method including UFLand MAG, with a first type flow connector connecting the UFL largeentity outlet and MAG inlet.

FIG. 44B illustrates first type sample processing method with a secondtype flow connector connecting the UFL large entity outlet and MAGinlet.

FIG. 44C illustrates first type sample processing method with a thirdtype flow connector connecting the UFL large entity outlet and MAGinlet.

FIG. 45A illustrates second type sample processing method including UFLand MAG, with a first type flow connector connecting the UFL smallentity outlet and MAG inlet.

FIG. 45B illustrates second type sample processing method with a secondtype flow connector connecting the UFL small entity outlet and MAGinlet.

FIG. 45C illustrates second type sample processing method with a thirdtype flow connector connecting the UFL small entity outlet and MAGinlet.

FIG. 46A illustrates third type sample processing method including MAGand UFL, with a first type flow connector connecting the MAG outlet andUFL entity fluid inlet.

FIG. 46B illustrates third type sample processing method with a secondtype flow connector connecting the MAG outlet and UFL entity fluidinlet.

FIG. 46C illustrates third type sample processing method with a thirdtype flow connector connecting the MAG outlet and UFL entity fluidinlet.

FIG. 47 illustrates fourth type sample processing method includingmultiple UFLs, a fourth type flow connector, and multiple MAGs.

FIG. 48 illustrates fifth type sample processing method includingmultiple UFLs, a fifth type flow connector, and multiple MAGs.

FIG. 49 illustrates fifth type sample processing method includingmultiple UFLs, a sixth type flow connector, and multiple MAGs.

FIG. 50 illustrates seventh type sample processing method includingmultiple MAGs, a fourth type flow connector, and multiple UFLs.

FIG. 51 illustrates eighth type sample processing method includingmultiple MAGs, a fifth type flow connector, and multiple UFLs.

FIG. 52 illustrates ninth type sample processing method includingmultiple MAGs, a sixth type flow connector, and multiple UFLs.

FIG. 53 illustrates tenth type sample processing method including one ormore of UFLs and MAGs, a fifth or a sixth type flow connector, anddifferent type of cell processing devices.

FIG. 54A illustrates eleventh type sample processing method including amulti-stage MAG process.

FIG. 54B illustrates twelfth type sample processing method including amulti-cycle MAG process.

FIG. 54C illustrates thirteenth type sample processing method includinga multi-stage UFL process.

FIG. 55A illustrates first example of closed and disposable fluidiclines for third type sample processing method.

FIG. 55B illustrates fluidic lines of FIG. 55A being connected to, orattached with, various fluidic devices to realize third type sampleprocessing method.

FIG. 56A illustrates second example of closed and disposable fluidiclines for third type sample processing method.

FIG. 56B illustrates fluidic lines of FIG. 56A being connected to, orattached with, various fluidic devices to realize third type sampleprocessing method.

FIG. 57A illustrates example of closed and disposable fluidic lines forfirst type sample processing method.

FIG. 57B illustrates fluidic lines of FIG. 57A being connected to, orattached with, various fluidic devices to realize first type sampleprocessing method.

FIG. 58A illustrates example of closed and disposable fluidic lines forsecond type sample processing method.

FIG. 58B illustrates fluidic lines of FIG. 58A being connected to, orattached with, various fluidic devices to realize second type sampleprocessing method.

FIG. 59A illustrates example of closed and disposable fluidic lines forsample processing through a single MAG.

FIG. 59B illustrates fluidic lines of FIG. 59A being connected to, orattached with, various fluidic devices to realize sample processingthrough a single MAG.

FIG. 60A illustrates example of closed and disposable fluidic lines forsample processing through a single UFL.

FIG. 60B illustrates fluidic lines of FIG. 60A being connected to, orattached with, various fluidic devices to realize sample processingthrough a single UFL.

FIG. 61A illustrates replacement of peristaltic pumps of FIG. 56B withpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 61B illustrates replacement of peristaltic pumps of FIG. 56B withvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 62A illustrates replacement of peristaltic pumps of FIG. 57B withpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 62B illustrates replacement of peristaltic pumps of FIG. 57B withvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 63A illustrates replacement of peristaltic pumps of FIG. 58B withpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 63B illustrates replacement of peristaltic pumps of FIG. 58B withvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 64A illustrates replacement of peristaltic pumps of FIG. 59B withpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 64B illustrates replacement of peristaltic pumps of FIG. 59B withvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 65A illustrates replacement of peristaltic pumps of FIG. 60B withpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 65B illustrates replacement of peristaltic pumps of FIG. 60B withvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 66 illustrates a first process flow to separate biological entitiesfrom peripheral blood using UFL and MAG.

FIG. 67 illustrates a second process flow to separate biologicalentities from peripheral blood using MAG.

FIG. 68 illustrates a third process flow to separate biological entitiesfrom peripheral blood using MAG.

FIG. 69 illustrates a fourth process flow to separate biologicalentities from peripheral blood using MAG.

FIG. 70 illustrates a fifth process flow to separate biological entitiesfrom tissue sample using UFL and MAG.

FIG. 71 illustrates a sixth process flow to separate biological entitiesfrom tissue sample using MAG.

FIG. 72 illustrates a seventh process flow to separate biologicalentities from surface swab sample using UFL and MAG.

FIG. 73 illustrates an eighth process flow to separate biologicalentities from surface swab sample using MAG.

FIG. 74 illustrates a ninth process flow to separate biological entitiesfrom solid sample using UFL and MAG.

FIG. 75 illustrates a tenth process flow to separate biological entitiesfrom solid sample using MAG.

FIG. 76A illustrates addition of both magnetic and fluorescent labelsinto fluid samples for specific binding to target cells or entities.

FIG. 76B illustrates incubation of both magnetic and fluorescent labelsat same time to form specific binding to target cells or entities.

FIG. 77A illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL before magnetic separation by MAG.

FIG. 77B illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL after magnetic separation by MAG.

FIG. 78A illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL before magneticseparation by MAG.

FIG. 78B illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL after magneticseparation by MAG.

FIG. 79 illustrates continued process of negative MAG sample through UFLand various cell processing devices and procedures.

FIG. 80 illustrates continued process of negative MAG sample through UFLand various particle or molecule processing devices.

FIG. 81 illustrates entities entity analysis of negative MAG sampleafter MAG separation into various analyzing devices.

FIG. 82 illustrates continued process of positive MAG sample through UFLand various cell processing devices and procedures.

FIG. 83 illustrates continued process of positive MAG sample through UFLand various particle or molecule processing devices.

FIG. 84 illustrates entities entity analysis of positive MAG sampleafter MAG separation into various analyzing devices.

FIG. 85A illustrates adding fluorescent labels to specifically bind totarget entities within negative MAG sample immediately after negativeMAG sample collection.

FIG. 85B illustrates adding fluorescent labels to specifically bind totarget entities within positive MAG sample immediately after positiveMAG sample collection.

FIG. 86A illustrates first example of cancer treatment.

FIG. 86B illustrates second example of cancer treatment.

FIG. 86C illustrates third example of cancer treatment.

FIG. 87 illustrates first method of tumor detection.

FIG. 88 illustrates second method of tumor detection.

FIG. 89 illustrates third method of tumor detection.

FIG. 90 illustrates embodiment of first process flow to obtain cell-freeplasma.

FIG. 91 illustrates embodiment of second process flow to obtaincell-free plasma.

FIG. 92 illustrates embodiment of third process flow to obtain cell-freeplasma.

FIG. 93 illustrates method of using tumor detector for anti-agingpurpose.

For purposes of clarity and brevity, like elements and components willbear the same designations and numbering throughout the Figures, whichare not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

While the current invention may be embodied in many different forms,designs or configurations, for the purpose of promoting an understandingof the principles of the invention, reference will be made to theembodiments illustrated in the drawings and specific language will beused to describe the same. It will nevertheless be understood that nolimitation or restriction of the scope of the invention is therebyintended. Any alterations and further implementations of the principlesof the invention as described herein are contemplated as would normallyoccur to one skilled in the art to which the invention relates.

Examples of biological entities described hereafter include: cell,bacteria, virus, molecule, particles including RNA and DNA, cellcluster, bacteria cluster, molecule cluster, and particle cluster. Largeentities and small entities refer to biological entities within the samefluid having relatively larger physical size and smaller physical size,respectively. In one embodiment, large entities include any of: cells,bacteria, cell cluster, bacteria cluster, particle cluster, entitiesbound with magnetic labels, and entities bound with optical label. Inanother embodiment, small entities include any of: molecules, particles,virus, cellular debris, non-bound free magnetic labels, and non-boundfree optical labels. In another embodiment, large entities have aphysical size larger than 1 micrometer (μm), and small entities have aphysical size less than 1 μm. In yet another embodiment, large entitieshave a physical size larger than 2 μm, and small entities have aphysical size less than 500 nanometer (nm). In yet another embodiment,large entities have a physical size larger than 5 μm, and small entitieshave a physical size less than 2 μm. Biological sample includes: blood,body fluid, tissue extracted from any part of the body, bone marrow,hair, nail, bone, tooth, liquid and solid from bodily discharge, orsurface swab from any part of body. Entity liquid, or fluid sample, orliquid sample, or sample solution, includes: biological sample in itsoriginal liquid form, biological entities being dissolved or dispersedin a buffer liquid, or biological sample after dissociation from itsoriginal biological sample non-liquid form and dispersed in a bufferfluid. Biological entities and biological sample may be obtained fromhuman or animal. Biological entities may also be obtained from plant andenvironment including air, water and soil. Entity fluid, or fluidsample, or sample may contain various types of magnetic or opticallabels, or one or more chemical reagents that may be added duringvarious steps within the embodiments of this invention. Sample flow rateis volume amount of a fluid sample flowing through a cross-section of achannel, or a fluidic part, or a fluidic path, in a unit time, wherevolume may be in unit of liter (l), milliliter (ml), microliter (μl), ornanoliter (nl), and unit time may be in unit of minute (min), second(s), millisecond (ms), microsecond (μs), or nanosecond (ns). Sample flowspeed is the distance that a free molecule or a free entity travelswithin a liquid sample in a channel, or a fluidic part, or a fluidicpath, in a unit time, where distance may be in unit of meter (m),centimeter (cm), millimeter (mm), or micrometer (μm). Separationefficiency is percentage of target entities within a liquid sample thatare successfully separated from the liquid sample by a method designedto separate the target entities. Buffer fluid is a fluid base wherebiological entities may be dissolved into, or dispersed into, withoutintroducing additional biological entities.

FIG. 4 shows a cross-sectional view of the first embodiment of amagnetic separation device (“MAG”) of the current invention. MAG 121 iscomposed of two magnetic field producing poles, pole 102 and pole 103.Each of the poles 102 and 103 is composed of soft magnetic material,which may include one or more elements of iron (Fe), cobalt (Co), nickel(Ni), iridium (Ir), manganese (Mn), neodymium (Nd), boron (B), samarium(Sm), aluminum (Al). Pole 102 has a magnetic flux collection end 1023and a tip end 1021, where shape of the pole 102 is converging from theflux collection end 1023 towards the tip end 1021. In FIG. 4 , fluxcollection end 1023 is a flat surface which is contacting, or inproximity to, the North Pole (“N”) surface of a permanent magnet 104.Permanent magnet 104 has a magnetization shown by arrow 1041 in FIG. 4which points from the South Pole (“S”) surface to the N surface of themagnet 104. Magnetization 1041 produces magnetic field in free space,which can be described as flux lines 1046 emitting from N surface andreturning to S surface of the magnet 104. Pole 102 flux collection end1023 being in contact with, or in close proximity to, the N surface ofmagnet 104 as shown in FIG. 4 . Due to the soft magnetic material ofpole 102, magnetic flux 1046 from the N surface of magnet 104 iscollected by the pole 102 and enters the body of the pole 102 throughflux collection end 1023. Due to the converging shape of the pole 102,the collected magnetic flux is mainly channeled within the soft magneticbody of the pole 102 and emitted from the tip end 1021 of pole 102. Saidclose proximity between flux collection end 1023 and N surface of magnet104 may be a gap distance in between surface of 1023 and N surface beingless than 1 mm. Tip end 1021 may have a much smaller surface area thanflux collection end 1023, which makes flux exiting the tip end 1021having a higher flux density than when flux 1045 is emitted by N surfaceof magnet 104, i.e. magnetic flux 1045 is concentrated and thus createsa local high magnetic field and high field gradient around the tip end1021. It is preferred that tip end 1021 of the pole 102 is as small aspossible, for example as a convergence point, to produce largest fluxconcentration for achieving highest magnetic field. However, inpractice, due to manufacturing process, tip end 1021 may have a curvedor domed shape, which would not affect the general concept of fluxconcentration by the tip end 1021. Pole 103 is similar to pole 102 inthat pole 103 has a larger flux collection end 1033 and a smaller tipend 1031, where flux collection end 1033 is in contact with, or in closeproximity to, S surface of permanent magnet 105. It is preferred thatpole 103 and magnet 105 are identical to pole 102 and magnet 104, butarranged to mirror pole 102 and magnet 104 around a center line 1050.Magnet 105 magnetization 1051 is opposite to magnetization 1041 ofmagnet 104. Magnetic flux 1047 collected by flux collection end 1033from S surface of pole 103 is opposite to that of pole 102, and fluxemitted from tip end 1031 of pole 103 is opposite to that of tip end1021. Thus, between the gap of tip ends 1021 and 1031, emitted flux canform closed loop and further enhance the magnetic field strength andfield gradient around the tip ends 1021 and 1031. Dashed lines 1045represent the flux emitted from tip end 1021 and returned to tip end1031. Flux lines 1045 closer to tip ends 1021 and 1031 being denserindicates stronger magnetic field and larger field gradient closer tothe gap area. As shown in FIG. 4 , top section of pole 102 is tilted tothe right side, while top section of pole 103 is tilted to the left.This tilted shape diverts the magnetic flux within poles 102 and 103away from bottom section of the poles and helps make the tip end 1021 ofpole 102 and tip end 1031 of pole 103 the closest spaced features of thepoles 102 and 103 to achieve high field in gap between tip ends 1021 and1031 while minimizing flux leakage between lower bodies of poles 102 and103. In FIG. 4 , the tilted top sections of the poles 102 and 103 form atriangle shape, or convex shape, top surface 1210 of the MAG 121, whichwill be referred to as “MAG wedge” 1210 of MAG 121 hereafter. Permanentmagnets 104 and 105 may be composed of any of, but not limited to, Nd,Fe, B, Co, Sm, Al, Ni, Sr, Ba, O, NdFeB, AlNiCo, SmCo, strontium ferrite(SrFeO), barium ferrite (BaFeO), cobalt ferrite (CoFeO).

FIG. 4 embodiment includes a rigid fixed shape channel 101. Channel 101has a channel wall enclosing a channel space 1013. The fluid sample mayflow through channel 101 in the channel space 1013 along the channel 101length direction that is perpendicular to the cross-section view of FIG.4 . Channel 101 has a top surface 1012 and a bottom surface 1011. Bottomsurface 1011 is formed in a shape conforming to the MAG wedge surface1210, such that when channel 101 is moved in direction 1014 to be incontact with the MAG 121 poles 102 and 103, bottom surface 1011 ofchannel 101 is in contact with MAG wedge surface 1210 with no or minimalgap in between bottom surface 1011 and MAG wedge surface 1210. Topsurface 1012 of channel 101 is preferred to be conformal to bottomsurface 1011 to produce a channel space 1013 with a shape that maximizesexposure of fluid sample flowing through channel 101 to the highestmagnetic field region of the MAG wedge gap field 1045.

In FIG. 4 embodiment, poles 102 and 103, magnets 104 and 105, andchannel 101 extend in the direction perpendicular to the cross-sectionview of FIG. 4 , which will be referred to as “length direction”hereafter. Fluid sample flows in the channel 101 and is contained inchannel space 1013 along the length direction. Channel 101 being a rigidand fixed shape channel, the wall thickness of channel 101 at surface1011 may be thinner than wall thickness at surface 1012, such thatchannel 101 mechanical robustness is maintained by the thicker wall atsurface 1012, and magnetic field effect on fluid sample is enhanced bythinner wall at surface 1011, allowing fluid sample being closer to theMAG wedge 1210 and tip ends 1021 and 1031. Channel 101 may be attachedto a non-magnetic channel holder 107 at the top surface 1012. Channelholder 107 may align channel 101 to MAG wedge 1210, move channel 101 toseparation position in contact with MAG 121, or lift channel 101 awayfrom MAG 121 after magnetic separation. Channel holder 107 may becomposed of any non-magnetic material including, but not limited to,metal, non-metal element, plastic, polymer, ceramic, rubber, silicon,and glass. In FIG. 4 , flux collection ends 1023 and 1033 of the softmagnetic poles 102 and 103 may also be referred to as base ends 1023 and1033.

Permanent magnets described in different embodiments of this invention,for example magnets 104 and 105 of FIG. 4 , may each have oppositemagnetization direction to that described in each of the figures andembodiments without affecting the designs, functions and processes ofthe embodiments.

FIG. 5 is the cross-sectional view of FIG. 4 first embodiment of MAGwith channel 101 in magnetic separation position. Channel 101 of FIG. 4moves along direction 1014 and comes into contact with MAG wedge 1210surface by bottom surface 1011. MAG 121 gap formed by tip ends 1021 and1031 is brought into contact with, or minimal distance to, wall ofchannel 101 and the fluid sample flowing in the channel 101. Channel 101“C” shape matching to the MAG wedge shape helps achieve largecross-sectional area of the channel space 1013 to maintain a high flowrate, and at the same time confines cells 10/30 in the fluid sampleflowing in channel 101 to a high field and high gradient region of theMAG 121 gap field as indicated by the field lines 1045. Compared toprior art of FIG. 3A and FIG. 3B, first embodiment MAG 121 does notintroduce foreign material into the channel 101 while achievingcomparable or higher magnetic field and field gradient on cells 10/30flowing through the channel 101. Removal of poles 102 and 103 togetherwith magnets 104 and 105 from channel 101 will eliminate fieldgeneration source and avoids limitation of prior art domain related cellloss. Compared to prior art of FIG. 3C, MAG wedge of FIG. 5 being incontact with the channel 101 wall brings highest achievable magneticfield and field gradient to the fluid sample in the channel 101 for amore efficient cell 10/30 separation. Channel 101 shape being conformalto MAG wedge shape allows channel 101 to have a large cross-sectionalsample flow area, while avoiding the deficiency of prior art that cells10/30 at top end of a circular channel experiencing much lower magneticfield than at lower end, which ultimately limits sample flow rate. Thus,sample flow rate in channel 101 can be higher than prior art whileachieving better magnetic separation efficiency.

FIG. 6 is same as FIG. 5 , except biological entities, or cells 10/30for simplicity of description, are included to describe magneticseparation by MAG 121 from a fluid sample 6. Fluid sample 6 carryingcells 10/30 is flown through channel 101 along length direction ofchannel 101 perpendicular to the FIG. 6 cross-sectional view. MAG 121gap magnetic field magnetizes the SPLs 2 attached to cells 10/30 andfield gradient pulls cells 10/30 from the fluid 6 towards the MAG wedgeto form conglomerate layer on the 1011 bottom surface of channel 101.Due to MAG 121 design and channel 101 shape, cells 10/30 close to topsurface 1012 experience magnetic field not significantly lower thanclose to bottom surface 1011, and distance for cells 10/30 to travelfrom top surface 1012 to conglomerate layer on bottom surface 1011 ismuch shorter than in prior art. These characteristics allow MAG 121 toresolve deficiencies of prior art.

FIG. 7 is a side view of FIG. 6 along direction 61 of FIG. 6 . Fluidsample 6 carrying cells 10/30 flows from left to right in the channel101 as indicated by arrow 1010. With the MAG 121 gap field, cells 10/30are separated from fluid 6 to form conglomerate on the channel wall ofbottom surface 1011. FIG. 7 shows that majority of the cells 10/30 areseparated from liquid 6 at the earlier section of the channel 101length, as indicated by the crowded population of cells 10/30. Ascertain tail population cells 10/30 may have comparatively smaller sizeSPLs 2 or fewer number of SPLs 2 bound to it surface, time required forsuch tail population cells to be pulled to bottom surface 1011 is longerthan nominal population when fluid 6 flows through channel 101. Thus,population of separated cells 10/30 will show density decrease frominlet towards outlet of channel 101.

FIG. 8A is a cross-sectional view of a second embodiment of MAG ofcurrent invention. MAG 122 in FIG. 8A is substantially similar to MAG121, except a soft magnetic shield 106 is attached to the S surface ofmagnet 104 and N surface of magnet 105. Magnetic flux from S surface ofmagnet 104 and N surface of magnet 105 forms closure path within thesoft magnetic shield 106. MAG 122, as compared to MAG 121, will haveless magnetic flux leakage outside of the MAG 122 structure. Themagnetic flux generated by magnets 104 and 105 are mainly confinedwithin the soft magnetic material body of poles 102 and 103, and shield106. MAG 122 is preferred in applications where magnetic interferencefrom MAG 122 to other surrounding instrument or equipment is desired tobe minimized.

FIG. 8B is a cross-sectional view of a third embodiment of MAG ofcurrent invention. Compared to MAG 121, MAG 123 of FIG. 8B incorporatesonly one permanent magnet 108, which is attached to both poles 102 and103. Flux from N surface of magnet 108 and flux from S surface ofmagnetic 108 is conducted by poles 102 and 103 to produce MAG 123 gapfield by tip ends 1021 and 1031. Compared to MAG 121, magnetic fluxgenerated by magnet 108 is mainly confined within the soft magneticmaterial body of poles 102 and 103, and MAG 123 is comparatively easierto assemble and produces less magnetic flux leakage.

FIG. 9 shows a cross-sectional view of the first embodiment MAG 121being used for magnetic separation in combination with a flexiblechannel 201. FIG. 9 is similar to FIG. 4 , except that the rigid channel101 is replaced with a flexible channel 201. Flexible channel 201 mayassume any shape, including a circular shape tubing form in itsnon-deformed state, but can be deformed into other shapes by externalforce. Wall material of channel 201 is deformable and may be composed ofany of, but not limited to, silicone, silicone rubber, rubber, PTFE,FEP, PFA, BPT, Vinyl, Polyimide, ADCF, PVC, HDPE, PEEK, LDPE,Polypropylene, polymer, thin metal or fiber mesh coated with polymerlayer. Flexible channel 201 is also shown in FIG. 9 to have a channelholder 107 attached to the back of channel 201. Channel holder 107 maybe composed of any non-magnetic material including, but not limited to,metal, non-metal element, plastic, polymer, ceramic, rubber, silicon,and glass. Channel 201 may attach to holder 107 through surface bonding,for example by gluing or injection molding, or via mechanical attachmentthrough components 1074 of FIG. 32 . Holder 107 has a bottom surface1070 in contact with the top surface of channel 201, where surface 1070is preferred to be substantially conformal to the MAG 121 wedge shape.In FIG. 9 , holder 107 aligns flexible channel 201 to MAG 121 wedge gapand moves channel 201 towards MAG wedge gap in direction 1014.

FIG. 10 illustrates the flexible channel 201 being pushed against theMAG wedge of MAG 121 by the channel holder 107. With pressure exerted bythe holder 107 on flexible channel 201 against the MAG wedge of MAG 121,channel 201 is deformed in FIG. 10 with bottom surface 2013 of channel201 becoming conformal and in surface contact with MAG wedge surface1210. Meanwhile, as holder 107 bottom surface 1070 may also be conformalto the MAG wedge shape, top surface 2012 of channel may also be moldedinto a shape that is substantially conformal to the MAG wedge. FIG. 10depicts the “separation position” of flexible channel 201 relative tothe MAG 121 during magnetic separation of cells 10/30 from sample fluid6. Shape of flexible channel 201 is substantially similar to channel 101of FIG. 5 and FIG. 6 , except such shape of channel 201 at separationposition is result of channel 201 self-aligning and self-conforming toMAG wedge without the need of a manufacturing process to achieve shapeof channel 101. Additionally, the flow space within channel 201 atseparation position may be adjusted to allow for larger or smallercross-sectional area of the flow space of channel 201, such thatoptimization of fluid sample 6 flow rate through channel 201 and cells10/30 magnetic separation efficiency may be optimized. The flow spaceadjustment may be achieved by changing the vertical distance 1071 fromthe holder 107 surface 1070 top point in contact with channel 201 topsurface 2012, to tip ends 1021 and 1031 or to an imaginary plane wheretip ends 1021 and 1031 reside. With a larger 1071 distance, flexiblechannel 201 is less deformed and a larger flow space is realized, whichallows for a slower flow speed at the same fluid flow rate. While with asmaller 1071 distance, flexible channel 201 has a smaller flow space buttop edge 2012 is also closer to the MAG wedge gap and tip ends 1021 and1031, which allows for higher magnetic field and faster separation ofcells 10/30. Thus optimization between flow rate and separationefficiency may be achieved by adjusting the distance 1071 for a givencombination of MAG 121 design and flexible channel 201. In oneembodiment, distance 1071 is more than 0 mm and less than or equal to 1mm. In another embodiment, distance 1071 is more than 1 mm and less thanor equal to 3 mm. In yet another embodiment, distance 1071 is more than3 mm and less than or equal to 5 mm. In yet another embodiment, distance1071 is more than 5 mm and less than or equal to 10 mm. In yet anotherembodiment, distance 1071 is more than 2 times and less than or equal to3 times of the wall thickness of flexible channel 201. In yet anotherembodiment, distance 1071 is more than 3 times and less than or equal to5 times of the wall thickness of flexible channel 201. In yet anotherembodiment, distance 1071 is more than 5 times and less than or equal to10 times of the wall thickness of flexible channel 201. Flexible channel201 at separation position functions similarly to channel 101 in FIG. 6. FIG. 10 also shows that during magnetic separation, cells 10/30 formconglomerate along channel 201 wall of lower surface 2013 directlyopposing the MAG wedge surface 1210. Thickness of channel 201 wall atbottom surface 2013 may be thinner than channel 201 wall at top surface2012.

FIG. 11 illustrates that after magnetic separation is completed in FIG.10 , the channel holder 107 moves away from the MAG 121 in direction1015, causing the flexible channel 201 to separate from MAG wedge of MAG121 to “lifted position.” Flexible channel 201 may also return to itsnon-deformed shape, for example circular tubing as shown in FIG. 11 .Magnetically separated cells 10/30 in FIG. 10 may retain theconglomerate form at the bottom surface of the flexible channel 201 inlifted position. After FIG. 11 lifted position of flexible channels 201is reached, dissociation procedures to break up the cells 10/30 in theconglomerate form within the flexible channel 201 may be performed, asdescribed in FIG. 22A through FIG. 30B. Flexible channel 201 returningto non-deformed shape, for example circular tubing of FIG. 11 , providesa larger cross-sectional area of the channel space 1013 as shown in FIG.11 than the separation position shown in FIG. 10 . Such larger channelspace 1013 may be preferred for easier dissociation of cells 10/30 fromthe conglomerate form. Additional buffer fluid may be injected into thechannel space 1013 of channel 201 in lifted position to assist channel201 returning to non-deformed shape.

MAG 121 in FIG. 9 through FIG. 11 may be replaced by MAG 122 or MAG 123without limitation on described methods and processes.

FIG. 12 illustrates cross-sectional view of the fourth embodiment of MAG124. MAG 124 has three soft magnetic poles 111, 112 and 113. Center pole111 is attached to N surface of permanent magnet 109 at a fluxcollection end 1112, similar to flux collection end 1023 of pole 102 inFIG. 4 . Flux 1048 from magnet 109 N surface is conducted by pole 111soft magnetic body and then emitted from a tip end 1111, which is muchsmaller in area size than flux collection end 1112 of pole 111 andfunctions similar to tip end 1021 of FIG. 4 to produce a local highfield around tip end 1111 by concentrating the magnetic flux conductedfrom magnet 109. Side poles 112 and 113 have flux collection ends 1122and 1132, respectively, which are attached to same top surface of a softmagnetic bottom shield 114. Bottom shield 114 is then attached to Ssurface of the permanent magnet 109. Thus the magnetic flux 1049 fromthe S surface of magnet 109 is conducted through the body of bottomshield 114 and divided between poles 112 and 113 and further conductedto the tip ends 1121 and 1131 of poles 112 and 113 respectively. Tip end1111 is formed in proximity to tip ends 1121 and 1131. In oneembodiment, tip end 1111 may recess from an imaginary plane, where tipends 1121 and 1131 reside, towards magnet 109 by an offset distancebetween 0 mm and 1 mm. In another embodiment, tip end 1111 may recessfrom an imaginary plane, where tip ends 1121 and 1131 reside, towardsmagnet 109 by an offset distance between 1 mm and 5 mm. In yet anotherembodiment, tip end 1111 may recess from an imaginary plane, where tipends 1121 and 1131 reside, towards magnet 109 by an offset distancebetween 5 mm and 10 mm. Tip end 1111 is preferred to be spaced equallyto tip ends 1121 and 1131. Top section of pole 112 is tilted to theright side, while top section of pole 113 is tilted to the left, similarto pole 102 and pole 103 of FIG. 4 . Such tilting is to increase gapbetween the main bodies of poles 112 and 113 to main body of pole 111 toreduce flux leakage such that flux concentration around tip ends 1111,1121 and 1131 is maximized. When flux is emitted from tip ends 1111,1121 and 1131, flux 1048 conducted by center pole 111 is opposite to theflux 1049 conducted by side poles 112 and 113, and the flux formsclosure between tip ends 1111 and 1121 and tip ends 1111 and 1131. Thus,the magnetic flux generated by N and S surface of magnet 109 isconducted within bodies of poles 111, 112, 113 and shield 114 withminimal leakage to outside of MAG 124 structure. Flux density is highestaround tip end 1111, with tip ends 1121 and 1131 also producing highflux density, which all indicate high magnetic field and field gradientaround tip ends 1111, 1121 and 1131. Compared to MAG 121, 122 and 123,MAG 124 has the advantage of more efficient flux closure within the MAG124 soft magnetic bodies with less leakage and thus higher flux densityaround tip end 1111 to produce higher magnetic field and field gradientin channel 301.

Channel 301 is a rigid channel similar to channel 101 of FIG. 4 , andhas a fixed shape similar to a rotated “D”. Channel 301 is shown to bein magnetic separation position in FIG. 12 , where tip ends 1111, 1121and 1131 may all be in contact with the curved bottom surface 3011 ofthe “D” shape channel 301 to provide highest possible magnetic field andfield gradient that MAG 124 can produce in the channel space throughwhich fluid sample flows in channel 301. In another embodiment, tip end1111 may be in contact with the surface 3011 and tip ends 1121 and 1131are not contacting surface 3011. Top surface 3012 of channel 301, in oneembodiment, may be on the imaginary plane where tip ends 1121 and 1131reside, and in another embodiment top surface 3012 may be above theimaginary plane by a distance in between 0 mm and 1 mm, and in yetanother embodiment top surface 3012 may be above the imaginary plane bya distance in between 1 mm and 5 mm. In one embodiment, channel 301 wallthickness at surface 3012 is thicker than wall thickness at surface3011. Channel 301 may be attached to a non-magnetic channel holder 110at the top surface 3012. Channel holder 110 may align channel 301 to MAGgap of MAG 124, move channel 301 to separation position in contact withMAG 124 pole 111 tip end 1111, or lift channel 301 away from MAG 124after magnetic separation.

FIG. 13 shows a cross-sectional view of the fourth embodiment MAG 124being used for magnetic separation in combination with the flexiblechannel 201, which is same as in FIG. 9 . Channel holder 110 may have adifferent shape than channel holder 107 of FIG. 9 . Before magneticseparation, channel holder 110 is attached to channel 201. Channelholder 110 aligns channel 201 to MAG gap of MAG 124, which is composedof tip ends 1111, 1121 and 1131 as in FIG. 12 , and moves channel 201into the MAG gap of MAG 124 in direction 1014.

FIG. 14 illustrates the flexible channel 201 in separation position inthe fourth embodiment MAG 124 with cells 10/30 being separated andforming conglomerate around bottom and side walls of the channel 201close to the tip ends 1111, 1121 and 1131. In FIG. 14 , flexible channel201 is deformed, similar to FIG. 10 , to conform to the MAG gapboundaries, which are mainly defined by the tip ends 1111, 1121 and1131. Shape of channel 201 may be different from channel 301 inseparation position due to flexible channel 201 conforming to the MAGgap boundaries under pressure from holder 110. Shape of channel 201 inFIG. 14 may provide higher liquid sample flow rate with higherseparation efficiency than channel 301. Distance 1071 between the lowersurface 1150 of holder 110 and tip end 1111 may be adjusted to optimizeflow rate in channel 201. Range of distance 1071 is same as 1071described in FIG. 10 .

FIG. 15A illustrates cross-sectional view of the fifth embodiment MAG125. MAG 125 is same as MAG 124, except the magnet 109 and bottom shield114 of MAG 124 of FIG. 12 are removed in MAG 125. Permanent magnets 115and 116 with opposing magnetizations 1151 and 1161 are placed in betweenpoles 111 and 112 and between poles 111 and 113, respectively, as shownin FIG. 15A. Magnetizations 1151 and 1161 are horizontal in FIG. 15A,which enables center pole 111 conducting N surface fluxes from bothmagnets 115 and 116, while side poles 112 and 113 conduct S surfacefluxes from magnet 115 and 116, respectively. Compared to MAG 124, MAG125 may produce higher field around tip ends 1111, 1121 and 1131 due totwo magnets 115 and 116 being used. MAG 125 may also be easier toassemble than MAG 124.

FIG. 15B illustrates cross-sectional view of the sixth embodiment MAG126. MAG 126 is same as MAG 124, except the side poles 112 and 113 areattached to S surfaces of permanent magnets 1092 and 1094, respectively,with magnetizations 1093 and 1095 being opposite to magnetization 1091of magnet 109. Bottom shield 114 is attached to both N surfaces ofmagnets 1092 and 1094 and S surface of magnet 109, thereby forminginternal flux closure in shield 114 between magnets 109, 1092 and 1094.Compared to MAG 124, MAG 126 may produce higher field around tip ends1111, 1121 and 1131 due to three magnets 109, 1092 and 1094 being usedin MAG 126.

FIG. 15C illustrates cross-sectional view of the seventh embodiment MAG127. MAG 127 is same as MAG 126 of FIG. 15B, except the bottom shield114 is removed.

FIG. 16 illustrates two of the third embodiment MAGs 123 being used formagnetic separation on a pair of flexible channels 201. The pair offlexible channels 201 are fixed on the same channel holder 1020 in FIG.16 . The top MAG 123 and bottom MAG 123 are substantially identical,with top MAG 123 being upside down vertically. MAG wedges of the top andbottom MAGs 123 are substantially aligned with center of top and bottomchannels 201. The magnets 108 of both top and bottom MAGs 123 may havesame magnetization direction, as the arrows within magnets 108 in FIG.16 indicate, such that the magnetic fields produced in the top andbottom channels 201 by the top MAG 123 and bottom MAG 123 duringmagnetic separation have same direction horizontal field component,which limits magnetic flux leakage between top MAG 123 soft magneticpoles and bottom MAG 123 soft magnetic poles.

FIG. 17 illustrates the two MAGs 123 of FIG. 16 being moved intoseparation position against the two flexible channels 201, which is sameprocess as in FIG. 10 . After reaching FIG. 17 separation position,fluid sample carrying cells 10/30 may flow through the channels 201 inlength direction perpendicular to the cross-section view to startmagnetic separation of cells 10/30 by top and bottom MAGs 123. Distance1071 between the holder 1021 surface contacting the channel 201 outeredge 2012 and MAG 123 tip ends 1021 and 1031, or the imaginary planewhere tip ends 1021 and 1031 reside, may be adjusted to optimize flowrate in each of the two channels 201. Range of adjustment of distance1071 is same as 1071 described in FIG. 10 .

MAG 123 in FIG. 16 and FIG. 17 may be replaced by MAG 121 or MAG 122,and channel 201 may also be replaced with channel 101.

FIG. 18 illustrates four of the fifth embodiment MAG 125 being used formagnetic separation on four flexible channels 201. The four flexiblechannels 201 are fixed on the same channel holder 1040 as shown in FIG.18 . The four MAGs 125 are substantially identical. MAG gaps of the fourMAGs 125 are substantially aligned with centers of the correspondingflexible channels 201. The permanent magnet arrangement within each MAG125 should be identical. For example, center pole of each of the fourMAGs 125 is attached to N surfaces of both magnets within eachrespective MAG 125, and side poles of each of the four MAGs 125 areattached to S surfaces of magnets within each MAG 125, as shown in FIG.18 . Thus, neighboring MAGs 125 closest to adjacent side poles have samemagnetic polarity, and leakage from side pole to side pole betweenneighboring MAGs 125 may be minimized or avoided. Additionally, fourMAGs used on four channels 201 in FIG. 18 is only shown in FIG. 18 as anexample of multiple channel process capability with a circular channelarrangement, where channels are positioned at center of the MAG 125circular array. Fewer or more MAGs 125 used on corresponding number ofchannels 201 may be achieved using FIG. 18 type circular arrangementwithout limitation. FIG. 18 multiple channel circular arrangement withMAG 125 is intrinsically more flexible than MAG 123 shown in FIG. 16 ,as two pole design of FIG. 16 MAG 123 may lead to magnetic flux leakagethrough the poles of neighboring MAGs 123 when number of MAGs 123 ismore than two.

FIG. 19 illustrates the four MAGs 125 of FIG. 18 being moved intoseparation position against the four flexible channels 201, which issame process as in FIG. 14 . After reaching FIG. 19 separation position,fluid sample carrying cells 10/30 may flow through the channels 201 inlength direction perpendicular to the view of FIG. 19 to start magneticseparation of cells 10/30 by the four MAGs 125. Similar to in FIG. 17 ,distance 1071 between the holder 1040 surface contacting the channel 201outer edge 2012 and MAG 125 center pole 111 tip end 1111 for eachchannel 201 and MAG 125 pair may be adjusted to optimize flow rate ineach of the four channels 201. Range of adjustment distance 1071 is sameas 1071 described in FIG. 10 .

MAG 125 in FIG. 18 and FIG. 19 may be replaced by MAG 124, MAG 126, orMAG 127. The channel 201 may also be replaced by channel 301.

FIG. 20A illustrates the sixth embodiment of MAG 128 with a rotated “D”shape rigid channel 320 in separation position. MAG 128 is similar toMAG 123, except that MAG wedge of MAG 123 is modified from a triangleshape to a flat top in MAG 128. MAG 128 pole 1022 is similar to pole 102of MAG 123, but with a flat top surface 1042 on pole 1022 instead of atip end on pole 102. Same flat top 1052 exists on pole 1032, which issimilar to pole 103 of MAG 123. Due to the flat top of the MAG wedge inMAG 128, rigid channel 320 may have a flat bottom surface 1062 matchingand in contact with the MAG wedge flat surface in separation position,to gain highest magnetic field and field gradient region from MAG 128.Channel 320 may be attached to a non-magnetic channel holder 1102 at thetop surface. Channel holder 1102 may align channel 320 to MAG wedge ofMAG 128, move channel 320 to separation position in contact with MAG 128poles 1022 and 1032 tip ends, or lift channel 320 away from MAG 128after magnetic separation.

FIG. 20B illustrates the sixth embodiment MAG 128 being used on aflexible channel 201, where channel 201 is attached to channel holder1102. Channel holder 1102 moves channel 201 towards MAG wedge of MAG 128along direction 1014.

FIG. 20C illustrates the sixth embodiment MAG 128 having the flexiblechannel 201 of FIG. 20B moved into separation position, with cells 10/30being separated from a liquid sample to form conglomerate at bottomsurface of channel 201, against the top flat surface of the MAG wedge ofMAG 128. Channel 201 is forced to form into a rotated “D” shape channelby holder 1102 pushing channel 201 against the flat top of MAG wedge ofMAG 128, where channel 201 shape in separation position is similar tochannel 320 of FIG. 20A. Distance 1071 between the holder 1102 bottomsurface 1062 contacting the channel 201 top edge 2012 and MAG 128 polesurfaces 1042 and 1052 may be adjusted to optimize flow rate in channel201. Range of adjustment distance 1071 is same as 1071 described in FIG.10 .

Magnet 108 of MAG 128 may be replaced by placement of magnets 104 and105 as in MAG 121, and by placement of magnets 104 and 105 and bottomshield 106 as in MAG 122.

FIG. 21A illustrates the seventh embodiment MAG 129 with a “V” shaperigid channel 330 in separation position. MAG 129 is different from MAG123 in pole shape, where pole 1024 and pole 1034 of MAG 129 have fluxconcentration tip ends 3301 and 3302 that form a “V” shaped notch,instead of the triangle wedge shape of the MAG 123. With the V shape MAGnotch of MAG 129, rigid channel 330 is also made into a V shape, withthe lower edges 3303 and 3304 making direct contact with the surfaces ofthe tip ends 3301 and 3302. Additionally, channel 330 may alsopreferably have a V shape notch recessed into the channel at the topedge 3305 following the V shape of the 3303 and 3304 edges, which helpsconfine fluid sample in the V shaped channel space 3306 to flow closerto the pole surfaces 3303 and 3004 that provide higher field and fieldgradient. Channel 330 may be attached to a non-magnetic channel holder1103 at the top surface 3305. Channel holder 1103 may align channel 330to MAG notch of MAG 129, move channel 330 to separation position incontact with poles 1024 and 1034 tip end surfaces, or lift channel 330away from MAG 129 after magnetic separation.

FIG. 21B illustrates the seventh embodiment MAG 129 being used with aflexible channel 201, where channel 201 is attached to channel holder1103 at the top edge of channel 201. Channel holder 1103 moves channel201 towards MAG 129 notch along direction 1014. Channel holder 1103 hasa triangle shape, where a convergence point of the triangle touches thechannel 201 top edge.

FIG. 21C illustrates the seventh embodiment MAG 129 with the flexiblechannel 201 of FIG. 21B moved into separation position, with cells 10/30being separated from a liquid sample to form conglomerate on bottomsurface of channel 201, against the top surfaces of the MAG notch of tipends 3301 and 3302 of MAG 129. Channel 201 is forced to form into a “V”shape channel by holder 1103. In FIG. 21C, holder 1103 forces channel201 against the MAG notch of MAG 129 with the lower convergence pointand deforms the top wall of the channel 201 downwards to move closer tothe tip ends 3301 and 3302. The same force also causes lower wall ofchannel 201 to conform to the MAG notch of MAG 129 and to make contactwith the tip ends 3301 and 3302 top surfaces 3303 and 3304. Thus,channel 201 shape in FIG. 21C in separation position shows V shapesimilar to channel 330 of FIG. 21A, which brings cells 10/30 in channelspace 3306 closer to high field and high gradient tip ends 3301 and 3302and tip surfaces 3303 and 3304. Vertical distance 1071 between theholder 1103 bottom convergence point contacting the channel 201 top edge2012 and MAG 129 tip ends 3301 and 3302, or an imaginary plane where tipends 3301 and 3302 reside, may be adjusted to optimize flow rate inchannel 201. Range of adjustment distance 1071 is same as 1071 describedin FIG. 10 .

Magnet 108 of MAG 129 may be replaced by placement of magnets 104 and105 as in MAG 121, and by placement of magnets 104 and 105 and bottomshield 106 as in MAG 122.

From FIG. 22A through FIG. 27D, various methods to demagnetize ordissociate magnetically separated cells 10/30 from conglomerate in MAGchannel will be described. For simplicity of description, flexiblechannel 201 is used. However, channels in FIG. 22A through FIG. 27D maybe labeled as “201/101”, indicating flexible channel 201 used fordescription may be replaced with rigid channel 101 without affecting thefunction and results of the described method. Also for the simplicity ofdescription, MAG 123 is used in FIG. 22A through FIG. 27D, while anyother MAG embodiment together with corresponding channel as described inprior figures may be used under same concepts without limitation

FIG. 22A is substantially similar to FIG. 10 , where channel 201 is atseparation position and cells 10/30 have been separated by magneticfield from MAG. In FIG. 22A, MAG 123 is used instead of MAG 121 of FIG.10 . Channel holder 1081 may be different from channel holder 107 ofFIG. 10 by having a top surface notch that allows the cells 10/30demagnetization by dissociation magnetic structure (“DMAG”), which ispermanent magnet 120 in FIG. 22A that is able to reach closer to thechannel 201/101 to provide sufficient field to demagnetize or dissociatecells 10/30 from the conglomerate in channel 201/101. Such notch ispreferred, but may not be required. DMAG magnet 120 is positioned awayfrom MAG 123 of FIG. 22A without affecting magnetic separation of cells10/30 by MAG 123. DMAG magnet 120 magnetization is labeled in verticaldirection 1201, but may also be in horizontal direction without causingfunctional difference. Channel 201/101 position relative to the MAG 123and DMAG 120 in FIG. 22A is “Position 1”.

FIG. 22B is similar to FIG. 11 , where channel holder 1081 moves channel201/101 away from MAG 123 and comes into contact with, or is in closeproximity to, DMAG magnet 120 at the top surface of holder 1081. Themagnet 120 may fit into the notch of holder 1081 to provide highestmagnetic field on cells 10/30 conglomerate in channel 201/101. Cells10/30 form conglomerate after magnetic separation by MAG and do notbreak free from the conglomerate automatically due to SPLs 2 on cells10/30 not self-demagnetizing when they are part of a conglomerate. Byremoving cells 10/30 gradually with magnetic field gradient from magnet120, for example cells 10/30 with higher magnetic moment SPL 2 thatrespond to weaker magnetic field from DMAG 120 faster, conglomerate mayreach to a critical volume that remaining cells 10/30 in theconglomerate do not see enough magneto-static field from other cells10/30 and will self-demagnetize into individual cells 10/30 due to theregained superparamagnetic nature of SPL 2. Therefore, to dissociatecells 10/30 from conglomerate, removing certain amount of cells 10/30,or breaking up the conglomerate from a continuous large piece intomultiple smaller pieces will help cells 10/30 to achieveself-demagnetization. Channel 201/101 position relative to the MAG 123and DMAG 120 in FIG. 22B is “Position 2”. Channel 201 compared tochannel 101 may have an advantage during cells 10/30 dissociation byDMAG magnet 120, as channel 201 provides a larger channel space thatallows farther separation between free cells 10/30 and conglomerate, orbetween broken-up conglomerate pieces, which helps to reducemagneto-static coupling and enhances self-demagnetization speed of SPLs2 on cells 10/30. For flexible channel 201, before Position 2 or inPosition 2, it is preferred to fill the channel 201 with additionalbuffer fluid to return the channel 201 to circular shape for largerchannel space.

FIG. 22C illustrates the cells 10/30 in the channel 201/101 of FIG. 22Bbeing dissociated from conglomerate by the DMAG magnet 120 in Position2.

FIG. 22D illustrates the channel holder 1081 moving channel 201/101 fromFIG. 22C DMAG Position 2 to a position, “Position 3”, between MAG 123and DMAG magnet 120. In Position 3, combined field on the cells 10/30within channel 201/101 may be the smallest, which may help SPL 2 toself-demagnetize. Channel 201/101 may be kept in Position 3 forextensive time to allow SPL 2 and cells 10/30 to fully self-demagnetizeand conglomerate to dissociate.

For an effective break up of conglomerate, mechanical agitations may beadded to the conglomerate by the magnetic forces exerted by MAG and DMAGmagnets. For example, channel holder 1081 may repeatedly move channel201/101 between Positions 1 and 2, or Positions 2 and 3, or Positions 1,2 and 3, such that alternating magnetic forces by MAG and DMAG may movewhole or part of the conglomerate in the channel space, thus helpingbreak up the conglomerate into smaller pieces or causing enough cells10/30 to break free from the conglomerate, which may self-dissociate.After conglomerate is sufficiently dissociated, free cells 10/30 may beflushed out of channel 201/101 in Position 3 or Position 2.

FIG. 23A illustrates that mechanical vibration may be applied to thechannel holder 1081 by a motor 130 when channel 201/101 is in Position 2of FIG. 22B or Position 3 of FIG. 22D. Such vibration may be transferredfrom holder 1081 through wall of channel 201/101 and into the fluidwithin the channel 201/101 to cause localized turbulence flow at variouslocations within the channel 201/101, which may help to mechanicallybreak up the conglomerate into small pieces to assist conglomeratedissociation.

FIG. 23B illustrates that ultrasound vibration by a piezoelectrictransducer (“PZT”) 131 may be applied to the channel holder 1081.Similar to FIG. 23A, ultrasound vibration may be transferred into thefluid within the channel 201/101 to cause localized high frequencyturbulence within the channel 201/101, which may help to mechanicallybreak up the conglomerate into small pieces to assist conglomeratedissociation.

FIG. 23C illustrates that mechanical vibration of FIG. 23A may beapplied to the channel 201/101 wall directly by motor 130.

FIG. 23D illustrates ultrasound vibration of FIG. 23B may be applied tothe channel 201/101 wall directly by PZT 131.

FIG. 23E is a side view of the channel 201/101 along the direction 61shown in FIG. 22D. Arrows 1030 represent alternating directions thatpulsed fluid flow may be applied to the channel liquid sample to producea flow jittering in the liquid within the channel 201/101, which mayalso produce local turbulence flow with fluid in channel 201/101 to helpmechanically break up the conglomerate into small pieces to assistconglomerate self-dissociation. FIG. 23E alternating pulsed flow may becombined with FIG. 23A through FIG. 23D vibration methods to apply tochannel 201/101 in Position 2 or Position 3 of FIG. 22B through FIG.22D.

When conglomerate in channel 201/101 is of large size, multiple roundsof cells 10/30 dissociation with FIG. 22B to FIG. 23E methods, andflushing of cells 10/30 out of channel 201/101, may be used. During eachflush, a certain part of cells 10/30 may be washed out of channel,making dissociation of remaining cells 10/30 still in the conglomeratein channel 201/101 easier in next round.

FIG. 24A is similar to FIG. 22B, where channel holder 1081 is incontact, or in close proximity to, DMAG magnet 120 after cells 10/30 aremagnetically separated by MAG 123. Different from that in FIG. 22B, DMAGmagnet 120 of FIG. 24A is positioned on the side of and away from MAG123, and holder 1081 is also rotated compared to FIG. 22B to fit its topsurface notch to the magnet 120. Placement of magnet 120 in FIG. 24A mayreduce magnetic field interference between MAG 123 and DMAG magnet 120.Channel 201/101 position relative to the MAG 123 and DMAG 120 in FIG.24A is “Position 12”.

FIG. 24B illustrates that after cells 10/30 are dissociated in Position12 of FIG. 24A, the channel 201/101 together with channel holder 1081 ofFIG. 24A are rotated away from magnet 120 of FIG. 24A into a positionbetween MAG 123 and DMAG magnet 120, where combined magnetic field fromMAG 123 and DMAG magnet 120 on channel 201/101 and cells 10/30 thereinis lowest, which is similar to Position 3 of FIG. 22D. Channel 201/101position relative to the MAG 123 and DMAG 120 in FIG. 24B is “Position13”.

FIG. 25A illustrates DMAG structure that is same as that in FIG. 22B,where DMAG structure includes only permanent magnet 120 withmagnetization 1201.

FIG. 25B illustrates DMAG structure that includes permanent magnet 120and a soft magnetic pole 1202 with convergence shape towards channel201/101. Soft magnetic pole 1202 convergence shape helps to concentratemagnetic flux from magnet 120 to produce higher field and high fieldgradient on cells 10/30 in channel 201/101 in Position 2 to moreeffectively demagnetize and dissociate the conglomerate of cells 10/30.

FIG. 25C illustrates DMAG structure that includes permanent magnet 120and a pair of soft magnetic poles 1203 and 1204. Magnetization 1201 ofmagnet 120 is in horizontal direction, and each of poles 1203 and 1204has an convergence shape pointing towards channel 201/101. Theconvergence ends of poles 1203 and 1204 form a DMAG gap sitting in, orin close proximity to, the channel holder 1081 top surface notch. Fluxfrom magnet 120 is conducted by the poles 1203 and 1204 and concentratedin the DMAG gap to produce high field and high field gradient on cells10/30 in channel 201/101 in Position 2 to more effectively demagnetizeand dissociate the conglomerate of cells 10/30.

FIG. 25D illustrates DMAG structure that includes an electromagnetincluding a soft magnetic core 1205 and coils 1206, where electriccurrent following in the coils 1206 may produce magnetization in core1205 in directions of 1207, and core 1205 functions like magnet 120 toproduct magnetic field on cells 10/30 in channel 201/101 in Position 2to demagnetize or dissociate the conglomerate of cells 10/30. Bychanging the electric current amplitude and direction in coils 1206,magnetic field from core 1205 on cells 10/30 may change strength anddirection. In one embodiment, DC current is applied to coils 1206. Inanother embodiment, AC current with alternating polarities is applied tocoils 1206. In yet another embodiment, current applied to coils 1206 isprogrammed to vary in amplitude, or in direction, or in frequency, or inamplitude ramp up or ramp down rate, to more effectively demagnetize anddissociate the conglomerate of cells 10/30.

FIG. 25E illustrates that motor 130 shown in FIG. 23A may producemechanical vibrations on DMAG structure of FIG. 25C. Such vibrations maybe transferred from DMAG structure to holder 1081 through DMAG structureto holder 1081 contact, and finally transferred to fluid in channel201/101, where DMAG structure can be changed to any of DMAG structuresdescribed in FIG. 25A through FIG. 25D.

FIG. 25F illustrates that PZT 131 shown in FIG. 23B may produceultrasound vibrations on DMAG structure of FIG. 25C. Such vibrations maybe transferred from DMAG structure to holder 1081 through DMAG structureto holder 1081 contact, and finally transferred to fluid in channel201/101, where DMAG structure can be changed to any of DMAG structuresdescribed in FIG. 25A through FIG. 25D.

To achieve demagnetization and dissociation of cells 10/30 fromconglomerate in channel 201/101, an alternative method as described inFIG. 26A through FIG. 26D may be used without using a DMAG structure,where the function of DMAG structure is achieved with the same MAG.

FIG. 26A is same as FIG. 22A, where channel 201/101 is in separationposition and cells 10/30 are separated by magnetic field of MAG 123 inchannel 201/101, except channel holder 1082 may not have the top surfacenotch as holder 1081. Channel 201/101 position relative to the MAG 123in FIG. 26A is “Position 21”.

FIG. 26B illustrates channel 201/101 of FIG. 26A is lifted from MAG 123to a lower field position, “Position 22”. In Position 22 channel 201/101may rotate around its center as indicated by arrow 210, preferable by180 degrees. Such rotation may require channel 201/101 not beingpermanently fixed to holder 1082

FIG. 26C illustrates channel 201/101 of FIG. 26B after rotation of 180degrees in Position 22. The cells 10/30 conglomerate formed on innerwall of channel 201/101 rotates together with channel wall to be at thetop end of the channel 201/101 relative to MAG 123.

FIG. 26D illustrates that channel 201/101 is moved from Position 22closer to MAG 123 to a Position 23 in between Position 21 and Position22, where magnetic field from MAG 123 on cells 10/30 is stronger thanPosition 22 but weaker than Position 21. Cells 10/30 in conglomerate attop end of channel 201/101 may then be pulled away by MAG 123 field fromconglomerate and demagnetization and dissociation of conglomerate maystart. The process of FIG. 26B through FIG. 26D may repeat multipletimes, where channel 201/101 may return to Position 22 from Position 23to perform another rotation and then move back to Position 23, untilcells 10/30 are sufficiently dissociated in channel 201/101. At end ofdemagnetization, cells 10/30 may be flushed out of channel 201/101preferably in Position 22. Mechanical vibrations and flow jittering asdescribed in FIG. 23C through FIG. 23E may be applied to channel 201/101in Position 22 and Position 23.

FIG. 27A is same as FIG. 26A, where channel 201/101 is in separationposition and cells 10/30 are separated by magnetic field of MAG 123.Channel 201/101 position relative to the MAG 123 is “Position 21”.Channel 201/101 is attached to holder 1082 in FIG. 27A.

FIG. 27B illustrates channel 201/101 of FIG. 27A being lifted from MAG123 to lower field Position 22 by holder 1082.

FIG. 27C illustrates that in Position 22, dissociation of cells 10/30 inchannel 201/101 may be achieved only through mechanical vibrationexerted by motor 130. FIG. 27C shows that motor 130 applies mechanicalvibration to holder 1082. Such vibration may be transferred from holder1082 through wall of channel 201/101 and into the fluid within thechannel 201/101 to cause localized turbulence flow at various locationswithin the channel 201/101, which may help to mechanically break up theconglomerate into small pieces to assist self-dissociation of cells10/30 conglomerate. Motor 130 may also exert vibration directly onchannel 201/101 as shown in FIG. 23C instead of through holder 1082.Alternating direction pulsed fluid flow as described in FIG. 23E may beapplied to the channel liquid sample to produce a flow jittering in theliquid within the channel 201/101 at the same time during motor 130vibration application.

FIG. 27D illustrates that in Position 22, dissociation of cells 10/30 inchannel 201/101 may be achieved primarily through ultrasound vibrationexerted by PZT 131. FIG. 27D shows that PZT 131 applies ultrasoundvibration to holder 1082. The ultrasound vibration may be transferredinto the fluid within the channel 201/101 to cause localized highfrequency turbulence within the channel 201/101, which may help tomechanically break up the conglomerate into small pieces to assistself-dissociation of cells 10/30 conglomerate. PZT 131 may also exertultrasound vibration directly on channel 201/101 as shown in FIG. 23D.Alternating direction pulsed fluid flow as described in FIG. 23E may beapplied to the channel liquid sample to produce a flow jittering in theliquid within the channel 201/101 at the same time during PZT 131ultrasound vibration application.

FIG. 28A through FIG. 30B describe embodiments of methods to assistcells 10/30 conglomerate dissociation by mechanical agitations, whichmay be applied to channel 201/101 in FIG. 27B in Position 22 and appliedto channel 201/101 in FIG. 22B through FIG. 22D, FIG. 23A through FIG.23D, FIG. 24A through FIG. 25F, FIG. 26B through FIG. 26D, FIG. 27Bthrough FIG. 27D.

FIG. 28A shows a side view of channel 201/101 and holder 1082 alongdirection 61 of FIG. 27B, where cells 10/30 are magnetically separatedby MAG field and form conglomerate on lower side of the channel 201/101wall. Channel mounts 1073 may be used to attach channel 201/101 tochannel holder 1082. Channel mounts 1073 may fix channel 201/101 atsections attached to mounts 1073 as anchors against channel 201/101deformation, compression or elongation during mechanical agitationprocess. Channel mounts 1073 may also perform a valve function thatcloses fluid flow into or out of flexible channel 201 section betweentwo channel mounts 1073 before mechanical agitation process of FIG. 28A,such that fluid enclosed in channel 201 may more efficiently producelocalized turbulence within the channel 201. FIG. 28A illustrates thatan externally applied force 300 may stretch or deform the channel201/101 in a direction away from the holder 1082, for exampleperpendicular to the channel 201/101 length direction. Such deformationor stretch of channel 201/101 builds up elastic energy in the channel201/101 wall material.

FIG. 28B illustrates that when force 300 of FIG. 28A is removed, elasticenergy built up in channel 201/101 wall acts to spring back channel201/101 towards its original non-deformed and non-stretched position.Depending on channel 201/101 wall material property, such spring backmay provide a transient turbulence flow at various locations within thechannel 201/101, which may help to mechanically break up the cells 10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30 conglomerate. After removal of force 300 and spring back of thechannel 201/101, alternating flow 1030 may be similarly applied as inFIG. 23E to assist dissociation process of conglomerate of cells 10/30,where valve function of channel mounts 1073 may be turned off to allowfluid flow within channel 201/101.

The deform/stretch and release process of the channel 201/101 asillustrated in FIG. 28A and FIG. 28B may be repeated as many times asneeded until conglomerate of cells 10/30 is sufficiently dissociated,which may then be flushed out of channel 201/101 by buffer fluid.

FIG. 29A illustrates an alternative method of mechanical agitation fromFIG. 28A. Every aspect is same as that in FIG. 28A, except that acompressive force 302 may be applied to compress channel 201 indirection perpendicular to the channel 201 length direction. Forexample, channel 201 is compressed against channel holder 1082 as shownin FIG. 29A. As liquid within channel 201 has limited compressibility,force 302 may cause channel 201/101 wall to expand in directionperpendicular to the view of FIG. 29A, i.e. in direction perpendicularto both channel length direction and force 302 direction. Such expansionof channel 201/101 wall will again build up elastic energy in thechannel 201 wall material.

FIG. 29B is same as FIG. 28B in every aspect, except that FIG. 29B isafter compressive force 302 of FIG. 29A is removed, and elastic energybuilt up in channel 201 wall acts to spring back channel 201 to itsoriginal non-compressed shape. Such spring back may provide a strongtransient turbulence flow at various locations within the channel 201,which may help to mechanically break up the cells 10/30 conglomerateinto smaller pieces to assist self-dissociation of cells 10/30conglomerate. After removal of force 302 and spring back of channelshape, alternating flow 1030 may be similarly applied as in FIG. 23E toassist dissociation process of conglomerate of cells 10/30, where valvefunction of channel mounts 1073 may be turned off.

The compression and release process of the channel 201 as illustrated inFIG. 29A and FIG. 29B may be repeated as many times as needed untilconglomerate of cells 10/30 are sufficiently dissociated, which may thenbe flushed out of channel 201.

FIG. 30A illustrates another alternative method of mechanical agitation.Every aspect is same as in FIG. 28A, except that rotational twistingforce 303 or 304 may be applied to channel 201 to twist channel 201along channel length direction, as shown in FIG. 30A. In one embodiment,only one of rotational force 303 or 304 is applied to one end of channel201. In another embodiment, both rotational forces 303 and force 304 areapplied to difference ends of the channel 201 in opposite rotationaldirections to cause the channel 201 to twist along channel lengthdirection. Such twist deformation of channel 201 will again build upelastic energy in the channel 201 wall material.

FIG. 30B is same as FIG. 28B in every aspect, except that FIG. 30B isafter rotational force 303 and force 304 of FIG. 30A are released, andelastic energy built up in channel 201 wall acts to spring back channel201 towards its original non-twisted shape. Such spring back may providea strong transient turbulence flow at various locations within thechannel 201, which may help mechanically break up the cells 10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30 conglomerate. After removal of forces 303 and 304, and spring backof channel 201 shape, alternating flow 1030 may be similarly applied asin FIG. 23E to assist dissociation process of conglomerate of cells10/30, where valve function of channel mounts 1073 may be turned off.

The twist and release process of the channel 201 as illustrated in FIG.30A and FIG. 30B may be repeated as many times as needed untilconglomerate of cells 10/30 is sufficiently dissociated, which may thenbe flushed out of channel 201 by buffer fluid.

Mechanical forces 300, 302, 303 and 304 may be applied by mechanicalstructures that are motorized and able to apply such forces repeatedlyto channel 201. Examples may include a flap for providing force 300, acompressor for provide force 302, and twisters for providing forces 303and 304.

FIG. 31 is a schematic diagram illustrating a method to use MAG toseparate biological entities conjugated with magnetic labels, forexample cells 10/30, from a fluid solution. MAG channel of FIG. 31 maybe any of channels 101, 201, 301, 320, or 330 described in any of thefigures accompanied this specification, and MAG of FIG. 31 may be any ofthe MAG 121, 122, 123, 124, 125, 126, 127, 128, or 129 described withthe corresponding channel in any of the said figures. Method of FIG. 31may include the following steps in sequence. In step 400, MAG channel ispositioned with its outside wall contacting MAG wedge surface or poletip ends, i.e. separation position of Position 1 or Position 21 as inFIG. 5 , FIG. 10 , FIG. 12 , FIG. 14 , FIG. 17 , FIG. 19 , FIG. 20A,FIG. 20C, FIG. 21A, FIG. 21C, FIG. 22A, FIG. 26A, FIG. 27A. In step 401,fluid sample is flowed through the MAG channel in separation position.Then in step 402, positive entities with SPLs 2 attached, for examplecells 10/30, and free SPLs 2 within the fluid sample are attracted bythe magnetic field of MAG and agglomerate at the MAG channel wallagainst the MAG wedge or MAG pole tip ends. Meanwhile, in step 402,negative entities without SPLs 2 attached pass through the MAG channelwithout being attracted. The negative entities may then be processeddirectly in subsequent procedures as shown by path 427, where subsequentprocedures may include entity analysis 407, for example processesincluded in FIG. 79 through FIG. 81 , or negative entities may be passedfor continued process 408, for example through a UFL device as shown inFIG. 46A through FIG. 46C, FIG. 50 through FIG. 52 , or through repeatedMAG process as in FIG. 54A and FIG. 54B. After step 402, in step 403,sample may be depleted at input of the MAG channel and magneticseparation of positive entities may be completed. In step 404, which isan optional step, buffer fluid may be flowed through MAG channel withMAG channel still at separation position to wash off any negativeentities without SPLs 2 but may have resided with the conglomerate ofpositive entities due to non-specific bindings. Then in step 405, MAGchannel may be moved away from MAG to dissociation position includingPosition 2 and Position 22 in FIG. 11 , FIG. 22B, FIG. 22D, FIG. 24B,FIG. 26B, FIG. 26D, FIG. 27B, and magnetic dissociation 451, as shown inFIG. 22A through FIG. 26D, or mechanical dissociation 452 as shown inFIG. 27C through FIG. 30B, or magnetic together with mechanicaldissociation 453 may be applied to the positive entities in MAG channel.In step 406, buffer fluid may be flowed through MAG channel to flush outdissociated positive entities. If positive entities are not completelydissociated, path 465 shows that repeated dissociation process 405 maybe applied to remaining positive entities in MAG channel after priorflush out step, until positive entities are sufficiently dissociated andflushed out of the MAG channel. In the case that fluid sample has alarge volume, fluid sample may be separated into multiple sub-volumes.After process of a sub-volume from step 400 to step 406, a nextsub-volume may be input into the MAG channel starting from step 400 forcontinued process as shown by path 461 until completion of the fluidsample of the large volume. After positive entities are collected afterstep 406, they may be processed in subsequent procedures as shown bypath 428, where subsequent procedures may include entity analysis 407 orcontinued process 408.

FIG. 32 illustrates a method to align channel 201/101 to MAG gap of MAG123 device. Precise alignment of channel 201/101 to MAG wedge or MAGpole tip ends is important as described in embodiments of thisinvention. In FIG. 32 , side fixtures 1074 may be used to align andposition channel 201/101 to designated locations on channel holder 1081or 1082. The fixtures 1074 may be fitted into a pre-defined slot, notch,clip or other physical features on the sides of the channel holder1081/1082. In one embodiment, channel 201/101 may be slightly stretchedin channel length direction. Thus channel 201/101 may have a reducedwidth 2011 in between the fixtures 1074. Such stretch helps to guaranteea straight channel which may be then aligned with a straight MAG wedgeof MAG 123. After channel 201/101 is attached to holder 1081/1082 byfixtures 1074, holder 1081/1082 may then move channel 201/101 toseparation position. Holder 1081/1082 may have a pre-determined physicalorientation with respect to MAG 123, for example a hinge, which alignschannel 201/101 to MAG wedge or MAG pole tip ends of MAG 123 precisely.Fixtures 1074 may be the same as channel mounts 1073 in FIG. 28A throughFIG. 30B.

FIG. 33A through FIG. 37 illustrate method to utilize peristaltic pumpsin embodiments of this invention.

FIG. 33A illustrates a typical peristaltic pump 500, which includes arotor 501, drivers 502 attached to the rotor 501, and pump tubing504/505, where tubing 504 is fluid incoming section and tubing 505 isfluid outgoing section of the same pump tubing. When rotor 501 rotatesin direction 503, drivers 502 will squeeze pump tubing and force fluidto move from incoming section 504 to outgoing section 505 in directions5041 and 5051, respectively. In the case when rotor rotates reversely todirection 503, fluid moves from outgoing section 505 to incoming section504 of the pump tubing. Connectors 506 and 507 may be optionalconnections to incoming fluid line and outgoing fluid line 508,respectively. Advantage for peristaltic pump is the tubing 504/505 maybe included as a continuous part of an enclosed fluid line as shown inFIG. 55A through FIG. 60B, which may be made disposable and single use,as well as sterile for clinical purpose. However, due to the spaceddrivers 502 along the circumference of the rotor 501, flow rate of fluidoutput from section 505 has pulsation behavior, where flow rateincreases and decreases with the movement of each of the driver 502.Such pulsation is not desired for MAG and UFL fluid driving. FIG. 33Ashows output section 505 outputs fluid through connector to channel 508.Channel 508 is preferred to be flexible tubing. Channel 508 may also bea section of channel 201. Flow limiter parts 509 and 510 functiontogether to effectively clamp onto the channel 508 to reduce the fluidflow rate passing through the limiter. With reduced flow rate throughthe limiter, continued fluid output from pump 500 section 505 into thechannel 508 will build up fluid pressure within channel 508. Due to theflexible nature of channel 508, channel 508 may enlarge its widthperpendicular to the channel length direction, and forms fluid reservoirwithin channel 508 with elastic stress built up in channel wall. Duringpulsation of output flow from pump 500, when 5051 flow rate increases,channel 508 width will increase to build up stress in 508 channel walland pressure within channel 508. The increased volume of channel 508absorbs most of the instantaneously incoming flow, while flow rate 520through limiters 509/510 into channel 201 shows smaller increase. When5051 flow rate decreases, build-in elastic stress in channel 508 walland fluid pressure in channel 508 continues to push fluid through thelimiters 509/510, and flow rate 520 shows smaller flow rate decrease.

FIG. 33B illustrates top-down view of the inner structure of first typeflow limiter 509 along the direction 63. FIG. 33B shows that flowlimiter 509 has a shaped trench 5011, which allows fluid to flow throughchannel 508 when limiters 509 and 510 clamp onto channel 508 as shown inFIG. 33A. Trench 5011 has entrance width 511 to incoming fluid and exitwidth 512 to channel 201, where width 511 may be larger than width 512.Decreasing trench 5011 width from 511 to 512 reduces the flow ratethrough the limiters 509/510. Flow limiter 510 may have same top downview and structure as limiter 509 when view in direction opposite to 63.

FIG. 33C illustrates a second type flow limiter in same view as FIG.33A. After limiters 509 and 510 clamp onto channel 508, flow limiters509/510 form an effective opening of 514 towards channel 508, andopening of 513 towards channel 201. Opening 513 may be smaller thanopening 514, which reduces flow rate through the limiters 509/510.

FIG. 34A is same as FIG. 33A except flow limiters 509/510 are disengagedfrom the flexible channel 508. Flow from pump 500 through channel 508and channel 201 is continuous without limiters 509/510 and there is noelastic stress built up in channel 508 wall.

FIG. 34B is a schematic illustration of fluid flow rate 520corresponding to FIG. 34A situation, which shows large pulsation in flowrate 520. FIG. 34B shows the example 520 flow rate value vs pump 500operation time from pumping start to pumping end. Value 521 illustratesthe high flow rate and value 522 illustrates low flow rate of thepulsation behavior.

FIG. 35A is same as FIG. 33A, where flow limiters 509/510 are clampedupon flow channel 508. Flow rate through the flow limiters 509/510 isreduced, and channel 508 has enlarged channel width with elastic stressbuilt up in channel 508 wall.

FIG. 35B is a schematic illustration of fluid flow rate 520corresponding to FIG. 35A situation, which shows pulsation reduction inflow rate 520 compared to FIG. 34B. Value 523 corresponds to value 521of FIG. 34B, and value 524 corresponds to value 522 of FIG. 34B. FIG.35B illustrates that limiters 509/510 effectively reduce 520 flow ratepulsation. Due to the channel 508 liquid pressure build up at the startof pumping, and channel 508 liquid pressure dissipation at end ofpumping, while limiters 509 and 510 are engaged, a flow rate ramp upslope 5221 after pump start and flow rate ramp down slope 5222 afterpump end may exist in FIG. 35B.

FIG. 36A and FIG. 36B illustrate method to use flow limiters 509/510 togenerate instantaneously high flow rate short pulse through channel 201for flushing out magnetically separated entities, for exampledissociated cells 10/30.

FIG. 36A illustrates FIG. 33A and FIG. 35A situation, where flowlimiters 509 and 510 are clamped onto the flexible channel 508 whilepump 500 pumps fluid into channel 508, where pressure is built up withinthe flexible channel 508, and elastic stress is built up in wall ofchannel 508. Line 525 represents a continuous channel 201 from after thelimiters 509/510 to channel 201 over MAG structure. Flow rate 5201represents averaged flow rate of flow rates 523 and 524 of FIG. 35B whenflow limiters 509 and 510 are engaged.

FIG. 36B illustrates that flow limiters 509 and 510 are disengaged fromthe flexible channel 508, similar to FIG. 34A situation, while pump 500still pumps fluid into channel 508, or immediately after pump 500 stopspumping and before pressure within channel 508 dissipates. Atdisengagement of limiters 509 and 510, liquid pressure in channel 508and elastic stress in wall of channel 508 produces an instant high speedfluid pulse flow 5202 into channel 201, which may flush the magneticallyseparated entities out of the channel 201. Such high speed short pulseflow 5202 may help to achieve complete flush out of cells 10/30 withsmall volume of fluid that is originally contained in channel 508 ofFIG. 36A. FIG. 36B also shows that a rigid cladding structure 1075 maybe put into contact with channel 201 to help reducing deformation offlexible channel 201 during the cells 10/30 flush out to maintain theflow speed in channel 201.

FIG. 37 is a schematic illustration of fluid flow rate pulse created bythe flow limiter operation of FIG. 36A to FIG. 36B, where 5201 is thefluid flow rate in channel 201 before limiters 509 and 510 are released,and 5202 is flow rate peak value after limiters 509 and 510 arereleased.

From FIG. 33A through FIG. 36B, channel 508 is a flexible channel, whilechannel 201 may be replaced by a rigid channel 101, 301, 320, or 330.

FIG. 38A through FIG. 43 describe various embodiments of micro-fluidicchip (“UFL”) and method of use.

FIG. 38A is a top-down view of a first UFL embodiment UFL 600, wheremicro-fluidic channels are formed as trenches into a substrate material611. UFL contains an entity fluid 6020 inlet 602, a buffer fluid 6040inlet 604, a main channel 601, a large entity 6070 outlet 607, and asmall entity 6090 outlet 609. Two side channels 603 connect inlet 602 tomain channel 601 from the two sides of the main channel 601. Inlet 604is directly connected to the main channel 601 at the center of the mainchannel 601. Main channel 601 connects to outlet 607 at the center ofthe main channel 601, and connects to outlet 609 from two sides of mainchannel 601 through two side channels 608. Entity fluid 6020 containsboth large entities 6070 and small entities 6090. Buffer fluid 6040 isfluid for providing UFL function but without biological entities. Largeentity 6070 fluid from outlet 607 contains mainly large entities 6070and buffer fluid 6040. Small entity 6090 fluid from outlet 609 containsmainly small entities 6090 and fluid of entity fluid 6020 and maycontain certain amount of buffer fluid 6040. During operation of UFL600, buffer fluid 6040 and entity fluid 6020 are simultaneously pumpedinto outlets 604 and 602, respectively. Buffer fluid 6040 flows alongcenter line of the main channel 601 and entity fluid flows close to thetwo sides of the main channel as laminar flow. Buffer fluid 6040 carrieslarge entities 6070 to exit outlet 607 and entity fluid carriesremaining small entities 6090 to exit outlet 609. Channel 601 issubstantially straight and linear along channel length direction frominlet 604 to outlet 607.

FIG. 38B is a cross-sectional view of a portion of the FIG. 38A UFL 600along direction 64, which includes entity fluid inlet 602, buffer fluidinlet 604, and part of the UFL main channel 601. FIG. 38B illustratesthat UFL 600 is composed of two components, substrate 611 and cover 610.Inlets 602 and 604, outlets 607 and 609, channels 601, 603 and 608 areformed in substrate 611 as trenches of same depth 627 and preferablyformed in a single step. In one embodiment, depth 627 is between 100 nmand 500 nm. In another embodiment, depth 627 is between 500 nm and 1 μm.In yet another embodiment, depth 627 is between 1 μm and 10 μm. In yetanother embodiment, depth 627 is between 10 μm and 100 μm. In yetanother embodiment, depth 627 is between 100 μm and 1 mm. Cover 610contains external access ports to inlets and outlets of UFL 600 to allowentity fluid 6020 and buffer fluid 6040 to enter inlets 602 and 604, andto allow large entity 6070 fluid and small entity 6090 fluid to exitoutlets 607 and 609. Inlets 602 and 604, outlets 607 and 609 are shownto be circular shape in FIG. 38A, but may be any other shape, includingellipse, square, rectangle, triangle, polygon, that is suitable forapplication. Access ports of cover 610 are clearances, i.e. holes,through cover 610 directly over the inlets and outlets 602, 604, 607 and609. FIG. 38B shows example of access ports 621 and 641 clearancesmatching to inlets 602 and 604 positions. After manufacture of the UFL600 substrate 611 with the trenches of inlets, outlets and channels, andcover 610 with the access ports, cover 610 is positioned over thesubstrate 611 to form enclosed channels 601, 603 and 608. Cover 610 maybond to substrate 611 through any of: (1) surface to surface Van derWaals force; (2) gluing; (3) ultrasound thermal melting when one or bothof substrate 611 and cover 610 are made of plastic or polymer material.Access port clearances of cover 610, for example clearances 621 and 641to inlets and outlets 602, 604, 607 and 609, are preferred to be smallerin size than the corresponding inlets and outlets, which allows forpositioning error during cover 610 to substrate 611 alignments withoutcausing function loss of UFL due to misalignment. Injectors 6021 and6041 then show example of possible external fluid injection to inlets ofUFL 600 through cover 610 access port clearances, where the injectors6021 and 6041 may have a larger nozzles size than the matching accessports 621 and 641 for managing positioning errors between injectors andaccess ports. FIG. 38B shows that entity fluid 6020 containing largeentities 612 and small entities 613, which may be injected by injector6021, passing through assess port 621 and into inlet 602 and passinginto main channel 601 as side laminar flows. Buffer fluid 6040 may beinjected by injector 6041, and passes through access port 641 and intoinlet 604 and then passes into main channel 601 as center laminar flow.

Substrate 601 may be composed of any of: glass, silicon,alumina-titanium carbide (AlTiC), plastic, polymer, ceramic, or metal,where metal may be composed of any one or any alloy of iron, nickel,chromium, platinum, tungsten, rhenium. In one embodiment, forming ofinlets, outlets and channels in substrate 611 includes the steps of: (1)providing a substrate 611 having one substantially flat surface; (2)forming etching mask on top of said flat surface; (3) etching ofsubstrate with a first etching method including: wet etch with fluidchemical, dry etch with chemical gas, plasma enhanced dry etch, sputteretch with ion plasma, and ion beam etch (IBE). Forming of etch mask ofstep (2), which may be composed of photo resist (PR), may includedeposition or spin coating of PR on said flat surface; exposure byoptical or ion/electron radiation with patterns of inlets, outlets andchannels; development of PR after said exposure, where remaining PR withsaid patterns serves as etch mask. Etch mask may also be made of a hardmask material that has lower etch rate than the substrate material underthe first etching method, and step (2) may include: deposition of a hardmask layer on said flat surface; deposition or spin coating PR layer onhard mask layer; exposure of said PR by optical or ion/electronradiation with patterns of inlets, outlets and channels, development ofPR after said optical exposure, where remaining PR with said patternsserves as etch mask for said hard mask; etching hard mask through with asecond etch method including any of: wet etch with fluid chemical, dryetch with chemical gas, plasma enhanced dry etch, sputter etch with ionbeam; removal of remaining PR layer. Second etch method and first etchmethod may be different in type, or different in chemistry.

In another embodiment, inlets, outlets and channels in substrate 611 maybe formed by thermal press involving the steps of using a heated stencilwith physical patterns of the inlets, outlets and channels to melt anddeform part of substrate 611 to construct the inlets, outlets andchannels, then cooling down substrate 611 and removing the stencil. Inthermal press, substrate material is preferred to be plastic or polymer.In yet another embodiment, inlets, outlets and channels in substrate 611may be formed by imprint, which involves the steps of using a stencilwith physical patterns of the inlets, outlets and channels to imprintinto a partially or completely melt substrate 611, and then cooling thesubstrate 611 and finally removing stencil, where cooled substrateretains the patterns of the inlets, outlets and channels transferredfrom stencil. In imprint, substrate material is preferred to be plasticor polymer. In another embodiment, inlets, outlets and channels areformed in substrate 611 by injection molding, where melted substrate 611material is injected into a mold cavity that defines substrate 611 bodywith engraved inlets, outlet and channels. Cover 610 may be composed ofa material similar to substrate 611 material. Access ports of cover 610may be similarly formed in cover 610 as the inlets, outlet and channelsbeing formed in substrate 611 as described above.

FIG. 38C is a schematic diagram illustrating a single fluidic pressurenode 615 created between two side walls of the UFL 600 channel 601 ofFIG. 38A by ultrasound vibration generated by a PZT 614. FIG. 38C is across-section view along direction 65 of FIG. 38A for part of the UFL600 including main channel 601, substrate 611, cover 610 and PZTtransducer 614 attached to the bottom of substrate 611. FIG. 38C showsthat after injection of entity fluid 6020 and buffer fluid 6040, entityfluid 6020 containing large entities 612 and small entities 613 mainlyflow along the edges of the channel 601 as laminar flow. AC voltage isapplied to PZT 614, where frequency (Fp) of AC voltage is preferred tobe at a frequency matching the PZT resonance frequency (Fr). PZT 614produces ultrasound vibrations in the substrate 611 at frequency Fp.Said ultrasound vibrations transfer to the fluid contained in channel601. Channel 601 has channel width 625 defined as the normal distancebetween the two side walls of channel 601. In one embodiment, width 625is between 100 nm and 1 μm. In another embodiment, width 625 is between1 μm and 10 μm. In yet another embodiment, width 625 is between 10 μmand 100 μm. In yet another embodiment, width 625 is between 100 μm and500 μm. In yet another embodiment, width 625 is between 500 μm and 5 mm.When channel width 625 is half wavelength, or an integer multiple ofhalf wavelength, of the ultrasound mode in the fluid within channel 601at frequency Fp, a standing wave may be present in between the two sidewalls of channel 601 as indicated by the dashed lines 626. FIG. 38Cshows that when channel width 625 is half wavelength of fluid ultrasoundmode at frequency Fp, a single fluidic pressure node 615 is formed alongthe center line of channel 601 in the direction of channel length, whichis perpendicular to the view of FIG. 38C. In another embodiment, channelwidth 625 is an integer multiplied by half wavelength of fluidultrasound mode at frequency Fp, where integer is larger than 1, andsaid integer number of fluidic pressure nodes may then be formed acrossthe width 625 with each node being a line along the direction of channellength. Presence of standing wave 626 and pressure node 615 exertsacoustic force, which is shown in FIG. 38D as arrows 628, on entities inthe entity fluid laminar flow along the side walls of channel 601 andcause large size entities 6070 to move close to center node 615 duringflowing through the channel 601. Said acoustic force 628 has thecharacteristics of: (1) largest amplitude close to channel 601 sidewalls with force directions pointing from the side walls towards thenode 615; (2) smallest force, or close to zero force, around node 615;(3) being linearly proportional to size of the entities; (4) being afunction of the density and compressibility of both the buffer fluid6040 and the entities. Due to these characteristics, with properoptimization of buffer fluid composition, buffer fluid 6040 laminar flowspeed, and entity fluid 6020 laminar flow speed, large entities 612 maybe optimized to preferably break the laminar flow barrier to enter thebuffer laminar flow due to a larger acoustic force acting on largeentities 612, and be concentrated around the center node 615.

FIG. 38D is a schematic diagram illustrating the fluid acoustic wave ofFIG. 38C causing larger size entities 612 to move into buffer fluidlaminar flow around center of the channel 601. When fluid within thechannel 601 exits the channel to outlets 607 and 609, channel 601 centersub-channel width 651 of FIG. 38A to outlet 607 may be much smaller thanthe width 625 of the channel 601, thus only allow large entities 612 atcenter flow within channel 601 to exit outlet 607 as large entity 6070fluid. While smaller entities 613 mainly in the close-to-side walllaminar flow exit channel 601 through side channels 608 to exit fromoutlet 609 as small entity 6090 fluid.

Frequency Fp of PZT 614 vibration in one embodiment is between 100 kHzand 500 Hz, between 500 kHz and 1 MHz in another embodiment, between 1MHz and 3 MHz in yet another embodiment, between 3 MHz and 10 MHz in yetanother embodiment, and between 10 MHz and 100 MHz in yet anotherembodiment. In FIG. 38C and FIG. 38D, PZT 614 may also be attached totop of cover 610 in FIG. 38C and FIG. 38D, and ultrasound vibrationsfrom PZT 614 is transferred from PZT 614 through cover 610 to fluidwithin channel 601, or through cover 601 to substrate 611 and then tothe fluid within channel 601.

FIG. 39 is a schematic diagram illustrating methods to use a UFL toseparate biological entities of different sizes, where UFL may be UFL600 from FIG. 38A or FIG. 40A, UFL 620, 630 and 640 from FIG. 41Athrough FIG. 43 . Sequential steps of 701 to 705 and 706 aresubstantially similar to that described in FIG. 38A, FIG. 38B, FIG. 38C,and FIG. 38D, except steps 703 and 704 refer to possibility of multiplepressure nodes, as shown in FIG. 41B and FIG. 42B. Step 707 entityanalysis can be performed on both the large entities 6070 and smallentities 6090, and may include processes 903, 904, 905, 906, 5824, 5825,5826 as described in FIG. 53 , FIG. 79 , FIG. 80 , FIG. 82 and FIG. 83on corresponding UFL output samples. Examples of continued process 708include further processing through a MAG device as shown in FIG. 44Athrough FIG. 45C, FIG. 47 through FIG. 49 , or through cascaded UFLprocess as in FIG. 54C.

FIG. 40A is a cross-sectional view of a portion of a UFL 650 similar toFIG. 38B. UFL 650 is identical to UFL 600 from a top-down view as inFIG. 38A, except that a uniform soft magnetic layer (“SML”) 616 isdeposited on top the substrate 611 of UFL 650, and patterned togetherwith the substrate 611 to form inlets 602 and 604, outlets 607 and 609,and channels 601, 603 and 608. SML 616 may be composed of at least oneelement from iron (Fe), cobalt (Co), and nickel (Ni). SML 616 thickness6164 is between 10 nm and 100 nm in one embodiment, between 100 nm and 1μm in another embodiment, between 1 μm and 10 μm in yet anotherembodiment, between 10 μm and 100 μm in yet another embodiment, between100 μm and 1 mm in yet another embodiment, and between 1 mm and 3 mm inyet another embodiment. Deposition of SML layer 616 on substrate 611 maybe accomplished by any of: electro-plating, vacuum plating,plasma-vapor-deposition (PVD), atomic layer deposition (ALD), chemicalvapor deposition (CVD). Etching of layer 616 together with substrate 611to form inlets 602 and 604, outlets 607 and 609, and channels 601, 603and 608 may be accomplished by any of: dry etch, plasma enhanced dryetch, ion plasma etch, and IBE. Layer 616 may be a continuous layeralong the channel 601 length direction and forms part of the side wallsof the channel 601.

FIG. 40B is similar to FIG. 38D and shows a schematic diagramillustrating that the large entities 612 are concentrated by acousticforce 628 to the channel 601 center around the pressure node 615 andsmall entities 613 mainly remain around the channel 601 side walls.Additionally, a magnetic field 617 is applied in-plane and inducesmagnetization 6162 in the SML layer 616. For the SML layer 616 locatedas part of the side walls of the channel 601, magnetization 6162produces local magnetic field 6163, which has strongest magnetic fieldstrength and field gradient close to the channel 601 side walls. Field6163 may help maintain magnetic small entities, for example free SPLs 2that are part of the entity fluid 6020 in positive sample after MAGseparation as shown in FIG. 82 and FIG. 83 , to stay within the laminarflow close to channel 601 side walls and output from outlet 609 of FIG.38A.

FIG. 40C shows that after etching of SML layer 616 together withsubstrate 611, and before cover 610 is attached to substrate 611, apassivation layer 6172 may be deposited covering exposed surfaces of SMLlayer 616 and substrate 611. Layer 6172 may help to isolate fluidreaction with material of SML layer 616. Layer 6172 may be depositedover the etched surfaces of SML 616 and substrate 611, preferablyconformably, by vacuum plating, electro-plating, PVD, ALD, CVD,molecular beam deposition (MBE), and diamond like carbon (DLC)deposition. Layer 6172 may be an oxide, or a nitride, or a carbide, ofany one or more elements of: Si, Ti, Ta, Fe, Al, W, Zr, Hf, V, Cu, Cr,Zn, Mg, Nb, Mo, Ni, Co, Fe, Ir, Mn, Ru, Pd, and C. Layer 6172 maycompose of at least one of. Si, Ti, Ta, Fe, Al, W, Zr, Hf, V, Cu, Cr,Zn, Mg, Nb, Mo, Ni, Co, Fe, Ir, Mn, Ru, Pd, and C. Layer 6172 may be aDLC layer. Thickness of layer 6172 may be between 1 nm and 10 nm in oneembodiment, between 10 nm and 100 nm in another embodiment, between 100nm and 10 μm in another embodiment, and between 10 μm and 100 μm inanother embodiment.

FIG. 41A is a top-down view of a second UFL embodiment UFL 620, which issame as FIG. 38A, except including a wider section 6012 of the mainchannel connecting between the inlet 604 and the narrower channelsection 601 of FIG. 38A. Slope 6016 represents a transition section 6016from wider section 6012 to narrow section 601. Channel sections 6012 and601 are substantially straight and linear along channel lengthdirection. Transition section 6016 may be a section of the main channel,where the main channel includes channel section 6012 connecting throughthe transition section 6016 to channel section 601. Transition section6016 functions to funnel fluid flow from wider section 6012 into thenarrower section 601. Channel wall of transition section 6016 mayintersect straight wall of wider section 6012 at a transition startpoint. Channel wall of transition section 6016 may intersect straightwall of narrower section 601 at a transition stop point. In oneembodiment, the channel shape of the transition section 6016 betweentransition start point and transition stop point may have a straightslope as shown in FIG. 41A. In another embodiment, the channel shape ofthe transition section 6016 between transition start point andtransition stop point may have a curvature, which may be tangential toone or both of channel wall of wider section 6012 and channel wall ofnarrower section 601.

FIG. 41B is a cross-sectional view of UFL 620 along direction 66 in FIG.41A, which is across the wider section 6012. Wider section 6012 has achannel width 6252, which corresponds to the full wavelength of theultrasound mode in the liquid within channel section 6012 at PZT 614operating frequency Fp as described in FIG. 38C, and is effectivelytwice the channel width 625 of channel 601 in FIG. 38C and FIG. 41C. Dueto channel width 6252 being equal to the full wavelength of ultrasoundmode at Fp, two pressure nodes may exist in channel section 6012.Acoustic force from the ultrasound mode may move and concentrate largeentities 612 at each of the two nodes from the channel wall entitylaminar flow.

FIG. 41C is a cross-sectional view of UFL 620 along direction 65 in FIG.41A, which is across the narrower section 601 and is identical to FIG.38D. After fluid within channel section 6012 flows through thetransition 6016 to channel section 601, single pressure node of channelsection 601 forces the large entities 612 to concentrate at channelsection 601 center, same as in FIG. 41C. Wider section 6012 provides afirst stage large entity 612 separation from small entities 613. Aftertransition section 6016, flow speeds of center buffer laminar flow andchannel side wall entity laminar flow increase to about twice the speedsof same flows in section 6012 due to the channel width reduction from6252 to 625. Channel section 601 provides a second stage large entityseparation from small entities, together with the increase flow speed inchannel section 601. Purity of large entities 612 in 6070 fluid outputfrom outlet 607, as well as purity of small entities 613 in 6090 fluidoutput from outlet 609, may be enhanced compared to UFL 600 of FIG. 38A.

FIG. 42A is a top-down view of a third UFL embodiment UFL 630, which isa further enhancement from the UFL 620 of FIG. 41A. Every aspect of FIG.42A is same as FIG. 41A, except that when compared to UFL 620 of FIG.41A, UFL 630 of FIG. 43A includes additional side channels 6013 thatconnect from around the transition section 6016 to side channels 608, orin another embodiment directly to the outlet 609, to divert side walllaminar flow of small entities 613 from wider section, which is alsoreferred to as first stage section 6012 as shown in FIG. 42B, directlyto output 6090 without entering narrower section, which is also referredto as second stage section 601. Channel sections 6012 and 601 aresubstantially straight and linear along channel length direction. In oneembodiment, side channels 6013 connect to first stage section 6012before the transition start point of section 6016 intersects section6012. In another embodiment, side channels 6013 connect to thetransition start point of section 6016 intersecting section 6012. In yetanother embodiment, side channels 6013 connect to a point within thetransition section 6016 between the transition start point of section6016 intersecting section 6012 and the transition stop point of section6016 intersecting section 601. In yet another embodiment, side channels6013 connect to the transition stop point of section 6016 intersectingsection 601. In yet another embodiment, side channels 6013 connect tothe second stage section 601 after the transition stop point of section6016 intersecting section 601.

FIG. 42B is a cross-sectional view of UFL 630 along direction 66 in FIG.42A, which is across the wider section 6012. FIG. 42B is identical toFIG. 41B.

FIG. 42C is cross-sectional view of UFL 630 along direction 65 in FIG.42A, which is across the narrower section 601 and side channels 6013.Compared to FIG. 41C, side channels 6013 connecting to around thetransition section 6016 of FIG. 42A contain mainly, or purely, smallentities 613. While the channel 601 of FIG. 42C includes large entities612 being separated and concentrated to channel 601 center pressure nodesimilar to FIG. 41C, small entities 613 around section 601 channel wallsare reduced in density when compared to FIG. 41C. Due to the pre-channelsection 601 small entity diversion by side channels 6013, UFL 630 mayhave an even higher purity of large entities 612 in fluid output 6070from outlet 607, as well as higher purity of small entities 613 in fluidoutput 6090 from outlet 609.

FIG. 43 is a top-down view of a fourth UFL embodiment UFL 640 having amultiple-stage UFL channel with sequential channel width reduction alongthe channel flow path. FIG. 43 shows a further enhancement in increasinglarge entity purity in fluid output 6070 and small entity purity influid output 6090. FIG. 43 shows that an additional wider width section6014 is added between inlet 604 and channel section 6012. Channel widthof section 6014 may be three times of the half wavelength of ultrasoundmode of the liquid flowing through the UFL 640 channel at PZT frequencyFp, and is one half wavelength wider than channel width 6252 of section6012. Channel width of section 6014 may also be wider than the channelwidth 6252 of next stage channel section 6012 by an integer multipliedby the half wavelength, where said integer is larger than one. Channelsection 6014 changes to reduced channel width section 6012 through atransition section 6017. Side channels 6015 connect from around thetransition section 6017 to side channels 6013, or 608, or directly tooutlet 609 to divert small entities 613 from channel side wall laminarflow of section 6014 from entering section 6012, thereby increasingpurity of large entity concentration in section 6012. Channel sections6014, 6012 and 601 are substantially straight and linear along channellength direction.

As a further extension from FIG. 43 , a multiple-stage UFL 640 may havemultiple channel sections along the UFL 640 channel flow path, whereeach earlier section of the UFL channel along the channel flow path mayhave a channel width that is wider than the immediate next sectionchannel width by an integer multiplied by a half wavelength ofultrasound mode in the fluid flow at the PZT frequency Fp, where saidinteger is equal to or larger than 1. Final channel section before flowexiting the outlets of the UFL 640 is preferred to have a channel widthequal to said half wavelength in one embodiment, but may also have achannel width that equals to an integer multiplied by said halfwavelength in another embodiment, where integer is larger than one. Sidechannels connecting to each of the transition areas between adjacentchannel sections divert small entities from the earlier channel in theentity laminar flow close to the earlier channel walls towards theoutlet 609 to reduce number of small entities entering into immediatenext stage channel section.

FIG. 44A through FIG. 65B illustrate various embodiments of method toutilize MAG and UFL devices to separate biological entities from anentity fluid. For simplicity of description UFL 600 of FIG. 38A and MAG123 with channel 201 are used in the figures for explanation. However,UFL 600 may be replaced with UFL 650, 630, 640 of FIG. 40A, FIG. 41A,FIG. 42A, FIG. 43 , while MAG 123 may be replaced with MAG 121, 122,124, 124,125, 126, 127, 128, 129 and corresponding channel types asdescribed in prior figures without limitation and without sacrifice ofperformance.

FIG. 44A illustrates first type sample processing method, wherebiological sample is first passed through UFL 600 and the large entityoutput 6070 of UFL 600 is then passed through channel 201 adapted to MAG123, with a first type flow connector 801 connecting the UFL 600 largeentity outlet 607 to MAG inlet flow as in step 401 of FIG. 31 or step708 of FIG. 39 . For the in series operation of UFL 600 and MAG 123devices, optimal flow rate for UFL channel 601 and optimal flow rate forMAG channel 201 may be different. Optimal flow rate for UFL channel 601acoustic force separation of large and small entities are determined bylaminar flow condition, and separation efficiency between large andsmall entities. Optimal flow rate for MAG 123 separation is determinedby the length of channel 201 and magnetic field force on magnetic labelsattached to the entities. Direct fluidically coupled flow from UFL 600outlet 607 to MAG channel 201 inlet will force the flow rate being thesame through UFL 600 channel and MAG channel 201, which may incurnegative impact on separation efficiency for either one or both of UFL600 and MAG 123. It is necessary to decouple the fluid flows through UFL600 and MAG 123 channel 201. Flow connector 801 serves to decouple flowrates of the UFL 600 and MAG 123. Output fluid 6070 is first injectedinto connector 801 through inlet 8011, and fluid in connector 801 isoutput through outlet 8012 as flow in steps 401/708 into inlet ofchannel 201 of MAG 123. Both UFL 600 and MAG 123 channel 201 may operateat their respective optimal flow rates. In one embodiment where MAG 123channel 201 optimal flow rate is larger than UFL 600 optimal flow rate,MAG 123 extracts fluid 401/708 from connector 801 faster than UFL 600injects fluid 6070 into the connector 801. A fluid level sensor 100 maybe attached to connector 801 to sense fluid level remaining in connector801. If fluid level drops below a low threshold, sensor 100 may signalMAG 123 to pause flow intake as in steps 401/708 to wait for connector801 internal liquid level to increase to a higher level before MAG 123may restart extracting fluid as in steps 401/708 from connector 801. Inanother embodiment where MAG 123 channel 201 optimal flow rate issmaller than UFL 600 optimal flow rate, MAG 123 extracts fluid as insteps 401/708 from connector 801 slower than UFL 600 injects fluid 6070into the connector 801. If fluid level increases above a high threshold,sensor 100 may signal UFL 600 to pause flow 6070 output to wait forconnector 801 internal liquid level to drop to another lower levelbefore UFL 600 may restart outputting fluid 6070 into connector 801.Flow connector 801 may have the design shown in FIG. 44A, where inlet8011 is at a higher vertical location than outlet 8012, and flow 6070enters connector 801 and accumulates at outlet 8012 at inside of 801 dueto gravity. Alternatively, liquid sample may be completely processedthrough UFL 600 first and stored in connector 801. MAG 123 then extractsfluid from connector 801 as input into the MAG 123 channel 201 andcompletes processing of all liquid sample from connector 801. Connector801 may be made as part of an enclosed fluidic line, where in the pathof flow 6070 from UFL 600 outlet 607 to inlet 8011 of connector 801, tooutlet 8012, to flow into inlet of channel 201 as in steps 401/708,fluid sample is not exposed to air, and is sterile.

FIG. 44B illustrates first type sample processing method of FIG. 44Ausing a second type flow connector 802 connecting the UFL 600 largeentity outlet 607 to MAG 123 channel 201 inlet. Connector 802 as shownin FIG. 44B takes the form similar to a vial. Flow 6070 enters connector802 through a short length inlet tube 8021 of connector 802 and drips tobottom of the connector 802 due to gravity. Flow as in steps 401/708 isextracted from the fluid at the bottom of the connector 802 by a longlength outlet tube 8022 to input of channel 201. Fluid level sensor 100may be attached to connector 802 to sense fluid level within connector802. UFL 600 and MAG 123 may both operate at their respective optimalflow rates, and fluid level sensor 100 may function to pause UFL 600operation or MAG 123 operation with the same method as described in FIG.44A. Alternatively, liquid sample may be completely processed throughUFL 600 and stored in connector 802. MAG 123 then extracts fluid fromconnector 802 as input into the MAG 123 channel 201 and completesprocessing of all liquid sample from connector 802. Connector 802 may bemade as part of an enclosed fluidic line similar to connector 801.

FIG. 44C illustrates first type sample processing method of FIG. 44Ausing a third type flow connector 803 connecting the UFL 600 largeentity outlet 607 to MAG 123 channel 201 inlet. Connector 803 as shownin FIG. 44C takes the form similar to a fluid bag or blood bag. Flow6070 enters connector 803 through a bottom inlet 8031 and fillsconnector 803 from bottom of the connector 803 due to gravity. Flow asin steps 401/708 is extracted from the fluid at the bottom of theconnector 803 through outlet 8032 to input of channel 201. Fluid levelsensor 100 may be attached to connector 803 to sense fluid level withinconnector 803. UFL 600 and MAG 123 may both operate at their respectiveoptimal flow rates, and fluid level sensor 100 may function to pause UFL600 operation or MAG 123 operation with the same method as described inFIG. 44A. Alternatively, liquid sample may be completely processedthrough UFL 600 and stored in connector 803. MAG 123 then extracts fluidfrom connector 803 as input into the MAG 123 channel 201 and completesprocessing of all liquid sample from connector 803. Connector 803 may bemade as part of an enclosed fluidic line similar to connector 801.

FIG. 45A illustrates second type sample processing method wherebiological sample is first passed through UFL 600 and the small entityoutput 6090 of UFL 600 is then passed through MAG 123, with first typeflow connector 801 connecting the UFL small entity 6090 outlet 609 toMAG 123 channel 201 inlet. FIG. 45A is identical to FIG. 44A in everyaspect except small entity flow 6090 from outlet 609 is injected intothe inlet 8011 of connector 801.

FIG. 45B illustrates second type sample processing method wherebiological sample is first passed through UFL 600 and the small entityoutput 6090 of UFL 600 is then passed through MAG 123, with second typeflow connector 802 connecting the UFL small entity 6090 outlet 609 toMAG 123 channel 201 inlet. FIG. 45B is identical to FIG. 44B in everyaspect except small entity flow 6090 from outlet 609 is injected intothe inlet 8021 of connector 802.

FIG. 45C illustrates second type sample processing method wherebiological sample is first passed through UFL 600 and the small entityoutput 6090 of UFL 600 is then passed through MAG 123, with third typeflow connector 803 connecting the UFL small entity 6090 outlet 609 toMAG 123 channel 201 inlet. FIG. 45C is identical to FIG. 44C in everyaspect except small entity flow 6090 from outlet 609 is injected intothe inlet 8031 of connector 803.

FIG. 46A illustrates third type sample processing method wherebiological sample is first passed through MAG 123 channel 201, andfollowing procedure 427 or 428 of FIG. 31 , the output of MAG 123channel 201 is then passed through UFL 600 as entity fluid 6020 intoinlet 602 as in step 408 of FIG. 31 , with first type flow connector 801connecting the MAG 123 channel 201 outlet to UFL 600 entity fluid 6020inlet 602. In FIG. 46A, output from MAG 123 can be either negativeentities that do not have attached SPL 2, or positive entities separatedby MAG 123 magnetic field and subsequently dissociated and flushed outof channel 201 as described in FIG. 31 . Similar to those in FIG. 44A,MAG 123 and UFL 600 may each operate with their respective optimal flowrate. Fluid level sensor 100 may be attached to connector 801 to sensefluid level remaining in connector 801. Fluid level sensor 100 operatessimilarly to that in FIG. 44A to sense fluid in connector 801, anddepending on the flow rate difference between MAG 123 and UFL 600, maypause MAG 123 or UFL 600 flow to maintain fluid level in connector 801above a low level or below a high level. Alternatively, liquid samplemay be completely processed through MAG 123 first and stored inconnector 801. UFL 600 then extracts fluid from connector 801 as inputinto the inlet 602 and completes processing of all liquid sample fromconnector 801. Connector 801 may be made as part of an enclosed fluidicline similar to that in FIG. 44A.

FIG. 46B is same as FIG. 46A in every aspect, except replacing connector801 with connector 802, where operation of connector 802 and attachedsensor 100 is same as that described in FIG. 44B.

FIG. 46C is same as FIG. 46A in every aspect, except replacing connector801 with connector 803, where operation of connector 803 and attachedsensor 100 is same as that described in FIG. 44C.

FIG. 47 illustrates fourth type sample processing method wherebiological sample is first passed through multiple UFLs 600. Outputfluids from the UFLs 600, which can be either large entities 6070 orsmall entities 6090, are then fed into inlets 8011 of a fourth type flowconnector 8010, and from connector 8010 outlets 8012 into the inlets ofchannels 201 of multiple MAGs 123. FIG. 47 is functionally similar toFIG. 44A and FIG. 45A. Connector 8010 is also functionally same asconnector 801, except inlets 8011 of connector 8010 accept multiplefluid outputs from multiple UFLs 600, and outlets 8012 of connector 8010output to inputs of multiple channels 201 of multiple MAGs 123.

FIG. 48 illustrates fifth type sample processing method where biologicalsample is first passed through multiple UFLs 600. Output fluids from theUFLs 600, which can be either large entities 6070 or small entities6090, are then fed into inlets 8021 of a fifth type flow connector 8020,and from connector 8020 outlets 8022 into the inlets of channels 201 ofmultiple MAGs 123. FIG. 48 is functionally similar to FIG. 44B and FIG.45B. Connector 8020 is also functionally same as connector 802, exceptinlets 8021 of connector 8020 accept multiple fluid outputs frommultiple UFLs 600, and outlets 8022 of connector 8020 output to inputsof multiple channels 201 of multiple MAGs 123.

FIG. 49 illustrates sixth type sample processing method where biologicalsample is first passed through multiple UFLs 600. Output fluids from theUFLs 600, which can be either large entities 6070 or small entities6090, are then fed into inlets 8031 of a sixth type flow connector 8030,and from connector 8030 outlets 8032 into the inlets of channels 201 ofmultiple MAGs 123. FIG. 49 is functionally similar to FIG. 44C and FIG.45C. Connector 8030 is also functionally same as connector 803, exceptinlets 8031 of connector 8030 accept multiple fluid outputs frommultiple UFLs 600, and outlets 8032 of connector 8030 output to inputsof multiple channels 201 of multiple MAGs 123.

In each of FIG. 47 , FIG. 48 , and FIG. 49 , in one embodiment, samebiological sample is divided and processed simultaneously throughmultiple UFLs 600. In another embodiment, each of the UFLs 600 processesa different biological sample. Output from each UFL 600, either largeentity 6070 fluid from outlet 607 or small entity fluid from outlet 609,shown as dashed lines in FIG. 47 , FIG. 48 and FIG. 49 , may beindividually input into the inlet 8011 of connector 8010 of FIG. 47 , orinto inlet 8021 of connector 8020 of FIG. 48 , or into inlet 8031 ofconnector 8030 of FIG. 49 , as shown by solid lines 6070/6090 in each ofFIG. 47 , FIG. 48 and FIG. 49 . From outlet 8012, 8022, 8032 of FIG. 47, FIG. 48 and FIG. 49 , respectively, following step 401 or 708, each ofthe MAGs 123 of FIG. 47 , FIG. 48 , or FIG. 49 may extract fluid samplefrom corresponding connector 8010, 8020, and 8030 into its correspondingchannel 201. Each of the UFLs 600 and each of the MAGs 123 of FIG. 47 ,FIG. 48 , or FIG. 49 may operate at its own respective optimal sampleflow rate, which may be different between different UFLs 600 anddifferent between different MAGs 123 within same figure. Due to theexistence of the connector 8010, 8020, and 8030, flow rate interferencesbetween the different UFLs 600 and MAGs 123 within each of FIG. 47 ,FIG. 48 and FIG. 49 are minimized or eliminated. Fluid level sensor 100may be attached to buffers 8010, 8020, and 8030 to sense fluid levelremaining in each of the flow connectors 8010, 8020, and 8030. Fluidlevel sensor 100 operates similarly to that in FIG. 44A through FIG. 44Cin sensing fluid in flow connectors 8010, 8020, and 8030, and dependingon the flow rate difference between MAGs 123 and UFLs 600 of eachfigure, may pause operation of one or more MAGs 123, or may pauseoperation of one or more UFLs 600 of each figure to maintain fluid levelin corresponding connector 8010, 8020, or 8030 to be above a low levelthreshold or below a high level threshold. Alternatively, liquid samplemay be completely processed through all UFLs 600 first and stored incorresponding connector 8010, 8020, or 8030 of each FIG. 47 , FIG. 48and FIG. 49 . MAGs 123 then extract fluid from corresponding connector8010, 8020, or 8030 of each figure and complete processing of all liquidsample from each corresponding connector 8010, 8020, or 8030. Connectors8010, 8020, and 8030 may each be made as part of a set of enclosedfluidic lines, which may include UFLs 600, channels 201 and connectionsfrom UFLs 600 to each connector 8010, 8020, 8030 and from each connector8010, 8020, or 8030 to channels 201, similar to that described in FIG.44A through FIG. 44C.

FIG. 50 illustrates seventh type sample processing method wherebiological sample is first passed through multiple MAGs 123. Outputfluids from the MAGs 123 channels 201 are then fed into inlets 8011 offlow connector 8010 of FIG. 47 , and from flow connector 8010 outlets8012 into the entity fluid inlets 602 of multiple UFLs 600.

FIG. 51 illustrates eighth type sample processing method wherebiological sample is first passed through multiple MAGs 123. Outputfluids from the MAGs 123 channels 201 are then fed into inlets 8021 offlow connector 8020 of FIG. 48 , and from flow connector 8020 outlets8022 into the entity fluid inlets 602 of multiple UFLs 600.

FIG. 52 illustrates ninth type sample processing method where biologicalsample is first passed through multiple MAGs 123. Output fluids from theMAGs 123 channels 201 are then fed into inlets 8031 of flow connector8030 of FIG. 49 , and from flow connector 8030 outlets 8032 into theentity fluid inlets 602 of multiple UFLs 600.

In each of FIG. 50 , FIG. 51 , and FIG. 52 , in one embodiment, samebiological sample is divided and processed simultaneously throughmultiple MAGs 123. In another embodiment, each of the MAGs 123 processesa different biological sample. Output from each MAG 123, either negativeentities following procedure 427, or positive entities followingprocedure 428, may be individually input into the inlet 8011 ofconnector 8010 of FIG. 50 , or into inlet 8021 of connector 8020 of FIG.51 , or into inlet 8031 of connector 8030 of FIG. 52 , as shown by solidlines 427/428 in each of FIG. 50 , FIG. 51 and FIG. 52 . From outlets8012, 8022, 8032 of FIG. 50 , FIG. 51 and FIG. 52 , respectively,following step 408, each of the UFLs 600 of FIG. 50 , FIG. 51 , or FIG.52 may extract fluid sample as entity fluid 6020 from correspondingconnector 8010, 8020, and 8030 into its corresponding entity inlet 602.Each of the UFLs 600 and each of the MAGs 123 of FIG. 50 , FIG. 51 , orFIG. 52 may operate at its own respective optimal sample flow rate,which may be different between different UFLs 600 and different betweendifferent MAGs 123 within same figure. Due to the existence of theconnectors 8010, 8020, and 8030, flow rate interferences between thedifferent UFLs 600 and MAGs 123 within each of FIG. 50 , FIG. 51 andFIG. 52 are minimized or eliminated. Fluid level sensor 100 may beattached to flow connectors 8010, 8020, and 8030 to sense fluid levelremaining in each of the flow connectors. Fluid level sensor 100operates similarly to that in FIG. 46A through FIG. 47C in sensing fluidin flow connectors 8010, 8020, and 8030, and depending on the flow ratedifference between MAGs 123 and UFLs 600 of each figure, may pauseoperation of one or more MAGs 123, or may pause operation of one or moreUFLs 600 of each figure to maintain fluid level in correspondingconnector 8010, 8020, or 8030 to be above a low level threshold or belowa high level threshold. Alternatively, liquid sample may be completelyprocessed through all MAGs 123 first and stored in correspondingconnector 8010, 8020, or 8030 of each of FIG. 50 , FIG. 51 and FIG. 52 .UFLs 600 then extract fluid from corresponding connector 8010, 8020, or8030 of each figure and complete processing of all liquid sample fromeach corresponding connector 8010, 8020, or 8030. Flow connectors 8010,8020, and 8030 may each be made as part of a set of enclosed fluidiclines, which may include UFLs 600, channels 201 and connections fromchannels 201 to each connector 8010, 8020, 8030 and from each connector8010, 8020, or 8030 to UFLs 600, similar to that described in FIG. 46Athrough FIG. 46C.

FIG. 53 illustrates tenth type sample processing method where biologicalsample after being passed through one or more of UFLs 600 or MAGs 123,output fluids from the UFLs 600 and MAGs 123 are fed into inlets of aflow connector 8020 or a flow connector 8030, and from the flowconnectors 8020 and 8030 outlets into different types of cell processingdevices. FIG. 53 shows example of liquid sample output from MAG 123channel 201, including negative entities following procedure 427 andpositive entities following procedure 428, may be injected to inlet 8021of connector 8020 or inlet 8031 of connector 8030 similar to that inFIG. 51 and FIG. 52 . Alternatively, UFL 600 large entity output 6070from outlet 607 or small entity output 6090 from outlet 609 may be alsoinjected into inlet 8021 of connector 8020 or inlet 8031 of connector8030 similar to that in FIG. 48 and FIG. 49 . After sample fluid iscompletely processed through UFL 600 or MAG 123, and injected into, andstored within, connector 8020 or connector 8030, entity analysis as instep 407 of FIG. 31 and step 707 of FIG. 39 may be performed by sendingsample fluid containing entities from connector 8020 or connector 8030to any of: cell counter 903, cell imager 904, flow cytometer or sorter905, and DNA or RNA sequencer 906. Entities may be further sent to DNAor RNA sequencer 906 after cell counter 903 as indicated by flow 936, orafter cell imager 904 as indicated by flow 946, or after flow cytometeror sorter 905 as indicated by flow 956. For sending the sample fluidfrom outlet 8022 of connector 8020, or from outlet 8032 of connector8030, pressurized chamber 800 may be used to contain the connector 8020or connector 8030 inside, and force sample fluid out of connector 8020or connector 8030 in a steady and continuous flow stream. Chamber 800may be a chamber filled with pressurized air inside. Connector 8020 ofvial type may have an additional air port 8023 open to chamber 800internal pressurized air to help pushing sample fluid out of connector8020. While connector 8030 may be in a flexible blood bag form, whichwhen under pressured air of chamber 800, will automatically deflate andforce sample liquid out through outlet 8032. To avoid back flow into UFL600 or MAG 123 channel 201, shut off valves 805 may be implemented onoutput lines from MAG 123 channel 201 and UFL 600 to connector 8020 orconnector 8030.

FIG. 54A illustrates eleventh type sample processing method wherebiological sample after being passed through a first MAG 123 channel 201during a magnetic separation may output negative entity fluid followingprocedure 427, or positive entity fluid following procedure 428, intoinlet of a second MAG 123 channel 201 input as in step 408 of acontinued process. FIG. 54A illustrates a multi-stage MAG process.

FIG. 54B illustrates twelfth type sample processing method where afterbiological sample being passed through MAG 123 for magnetic separation,output fluid from MAG 123 channel 201, containing either negativeentities or position entities, may be diverted through a T-connector 912into flow 913. Flow 913 may then be re-input back into the input of thechannel 201 of MAG 123 for another round of magnetic separation throughT-connector 911. T-connector 911 allows initial fluid sample input as instep 401 and recycled flow 913 input to channel 201. T-connector 912allows output from channel 201 into recycled flow 913 or output from MAG123 as described in procedures 427 and 428. In one embodiment, recycledflow 913 contains negative entities. Repeated magnetic separation inFIG. 54B helps to achieve complete depletion of all magnetic entities inthe negative entity flow before continuing to 427/428 procedure. Inanother embodiment, recycled flow 913 contains positive entities afterdissociation. Repeated process as shown in FIG. 54B helps to increasepurity in positive magnetic entities to allow wash off of non-magneticentities that may be in the conglomerate by non-specific bindings. FIG.54B illustrates the use of the same MAG 123 for a multi-cycle MAGprocess.

FIG. 54C illustrates thirteenth type sample processing method wherebiological sample after being passed through a first UFL 600, outputfluids from first UFL 600, for example large entity fluid 6070 fromoutlet 607 or small entity fluid 6090 from outlet 609, may be passedinto entity fluid inlets 602 of one or more subsequent UFLs 600 as amulti-stage UFL process.

FIG. 55A illustrates first embodiment of closed and disposable fluidiclines for third type sample processing method shown in FIG. 46A, whereconnector 801 may be replaced with connector 802 or connector 803without limitation. Input line 923 may connect to a sample liquidcontainer. Input line 924 may connect to a MAG buffer container. Inputline 923 and input line 924 are connected through a T-connector 921 tothe inlet of the first pump tubing 504/505 that may be mounted onto aperistaltic pump. First pump tubing 504/505 outlet connects to channel201 which may be used as part of MAG 123. Output of channel 201 connectsto T-connector 922, which connects to output line 925 and output line926. Output line 925 may connect to an MAG output sample container andoutput line 926 connects to inlet of connector 801. In one embodiment,output line 925 may output negative entities to said MAG output samplecontainer, and output line 926 may output positive entities to connector801. In another embodiment, output line 925 may output positive entitiesto said MAG output sample container, and output line 926 may outputnegative entities to connector 801. Connector 801 outlet connects toinput line 9271 of a second pump tubing 504/505. Said second pump tubing504/505 outlet then connects to UFL 600 sample input line 6020. Inputline 9272 may connect to UFL buffer container and connects to inlet of athird pump tubing 504/505. Said third pump tubing 504/505 outlet thenconnects to UFL 600 buffer input line 6040. UFL 600 large entity 6070output line may connect to a large entity sample container. UFL 600small entity 6090 output line may connect to a small entity samplecontainer. FIG. 55A illustrates that besides the input and output lines923, 924, 925, 9272, 6070, and 6090 that connect to external containersand entire fluid path from sample liquid input to line 923 and fromsample output to lines 925, 6070, 6090, all pumps, MAG 123 and otherfluidic line components will be externally attached to the lines of FIG.55A. Thus, lines of FIG. 55A are internally enclosed, suitable forsingle use disposable purpose and sterile applications.

FIG. 55B illustrates fluidic lines of FIG. 55A being connected to, orattached with, various fluidic components. Input line 923 connects to aliquid sample container 928 in blood bag form. Input line 924 connectsto buffer container 929. Valves 935 and 936 are attached to lines 923and 924 to control either sample liquid from bag 928 or buffer fromcontainer 929 flowed through T-connector 921 into first pump tubing504/505. First, second and third pump tubings 504/505 are each installedonto a peristaltic pump 500. Three pumps 500 operate to pump eithersample fluid or buffer fluid into MAG 123 and UFL 600. A flow limiter509/510 may be attached to the output line from each pump 500, includinglines 201, 6020, 6040 to reduce flow rate pulsation from the pumps 500.Channel line 201 is mounted onto MAG 123. Output line 925 connects toMAG output sample container 934. Valve 940 is attached to line 925 andvalve 937 is attached to line 926, which control negative entities orpositive entities from MAG 123 going into either container 934 or theconnector 801 through the T-connector 922. Valves 940 and 937 may bothshut down the flow in lines 925 and 926 duringdemagnetization/dissociation process of MAG 123. Input line 9272 mayconnect to UFL buffer container 931. UFL output line 6070 connects tolarge entity container 932 and output line 6090 connects to small entitycontainer 933. Adjustable valves 939 and 938 may be attached to thelines 6070 and 6090 to adjust the flow rate within each of lines 6070and 6090, which in turn controls the laminar flow speed in UFL channelfor channel center buffer flow and channel edge entity sample flow.

FIG. 56A illustrates second embodiment of closed and disposable fluidiclines for third type sample processing method shown in FIG. 46A. FIG.56A is identical to FIG. 55A, except the output line 925 is connected toa MAG sample container 934, UFL output line 6070 is connected to a largeentity container 932, and UFL output line 6090 is attached to a smallentity container 933. FIG. 56A illustrates that containers 934, 932, 933are in the form of blood bags. Bags 932, 933, 934 as part of theenclosed lines of FIG. 56A may be disposable and made sterile, and mayalso be separated from the lines after separation process in steps 407and 408 of FIG. 31 , or steps 707 and 708 of FIG. 39 .

FIG. 56B describes the process of connecting sample container 928,buffer container 929, buffer container 931 to the lines 923, 924 and9272, respectively, same as in FIG. 55B. Containers 928, 929, 931 are inblood bag form. Also same as described in FIG. 55B, three pump tubings504/505 are installed on the three peristaltic pumps 500, valves 935,936, 940, 937, 939, 938, are each attached to the corresponding line,and flow limiter 509/510 may be attached to output line of each pump500, same as in FIG. 55B.

FIG. 57A illustrates embodiment of closed and disposable fluidic linesfor first type sample processing method shown in FIG. 44A, whereconnector 801 may be replaced with connector 802 or connector 803without limitation. Input line 9271 may connect to a UFL sample liquidcontainer, and also connects to inlet of a first pump tubing 504/505,which further connect to entity input line 6020 of UFL 600. Input line9272 may connect to a UFL buffer container, and also connects to inletof a second pump tubing 504/505, which further connect to buffer inputline 6040 of UFL 600. UFL 600 large entity output line 6070 connects toinlet of connector 801. UFL 600 small entity output line 6090 mayconnect to a small entity container. Outlet of connector 801 connects toMAG sample input line 923. MAG buffer input line 924 may connect to aMAG buffer container. Input lines 923 and 924 are connected through aT-connector 921 to the inlet of the third pump tubing 504/505. Thirdpump tubing 504/505 outlet connects to channel 201, which may be used aspart of MAG 123. Output of channel 201 connects to T-connector 922,which connects to output line 925 and output line 926. Output lines 925and 926 may each connect to an MAG output sample container. In oneembodiment, output line 925 may output negative entities to a first MAGoutput sample container, and output line 926 may output positiveentities to a second MAG output sample container. FIG. 57A illustratesthat besides the input and output lines 9271, 9272, 924, 6090, 925, and926 that connect to external containers and entire fluid path from UFLsample and UFL buffer to input lines 9271 and 9272 and from sampleoutput to lines 6090, 925 and 926, all pumps, MAG 123 and other fluidicline components will be externally attached to the lines of FIG. 57A.Thus, lines of FIG. 57A are internally enclosed, suitable for single usedisposable purpose and sterile applications.

FIG. 57B illustrates fluidic lines of FIG. 57A being connected to, orattached with, various fluidic components. First, second and third pumptubings 504/505 are each installed onto a peristaltic pump 500. Threepumps 500 operate to pump either sample fluid or buffer fluid into MAG123 and UFL 600. A flow limiter 509/510 may be attached to the outputline from each pump 500, including lines 201, 6020, 6040, to reduce flowrate pulsation from the pump 500. Input line 9271 connects to a liquidsample container 928 in blood bag form. Input line 9272 connects to UFLbuffer container 931 also in blood bag form. UFL output line 6090connects to small entity container 933 in blood bag form. Adjustablevalves 939 and 938 may be attached to the lines 6070 and 6090 to adjustthe flow rate within each of lines 6070 and 6090, which in turn controlsthe laminar flow speed in UFL 600 channel for channel center buffer flowand channel edge entity sample flow. Input line 924 connects to MAGbuffer container 929. Valves 935 and 936 are attached to lines 923 and924 to control either sample liquid from connector 801 or buffer fluidfrom container 929 flowed through T-connector 921 into third pump tubing504/505. Channel line 201 is mounted onto MAG 123. Output line 925connects to first MAG output sample container 934. Output line 926connects to second MAG output sample container 9342. Valve 940 isattached to line 925 and valve 937 is attached to line 926, whichcontrol negative entities and positive entities from MAG 123 going intoeither container 934 or container 9342 through the T-connector 922.Valves 940 and 937 may both shut down the flow in lines 925 and 926during demagnetization/dissociation process of MAG 123.

FIG. 58A illustrates embodiment of closed and disposable fluidic linesfor second type sample processing method shown in FIG. 45A. FIG. 58A isidentical to FIG. 57A in every aspect, except the UFL 600 small entityoutput line 6090 connects to the inlet of the connector 801 instead ofthe output line 6070 as in FIG. 57A. Large entity output line 6070 ofFIG. 58A may connect to a large entity container.

FIG. 58B illustrates fluidic lines of FIG. 58A being connected to, orattached with, various fluidic components. FIG. 58B is identical to FIG.57B in every aspect, except the UFL 600 small entity output line 6090connects to the inlet of the connector 801 instead of the output line6070 as shown in FIG. 57B. Large entity output line 6070 of FIG. 58Bconnects to a large entity container 932 in blood bag form.

FIG. 59A illustrates embodiment of closed and disposable fluidic linesfor sample processing through a single MAG. Input line 923 may connectto a sample liquid container. Input line 924 may connect to a MAG buffercontainer. Input line 923 and input line 924 are connected through aT-connector 921 to the inlet of the pump tubing 504/505 that may bemounted onto a peristaltic pump. Pump tubing 504/505 outlet connects tochannel 201 which may be used as part of MAG 123. Output of channel 201connects to T-connector 922, which connects to output line 925 andoutput line 926. Output lines 925 and 926 may each connect to an MAGoutput sample container.

FIG. 59B illustrates fluidic lines of FIG. 59A being connected to, orattached with, various fluidic components. Input line 923 connects to aliquid sample container 928. Input line 924 connects to buffer container929. Valves 935 and 936 are attached to lines 923 and 924 to controleither sample liquid from bag 928 or buffer from container 929 flowedthrough T-connector 921 into first tubing 504/505. Pump tubing 504/505is installed into a peristaltic pump 500. Pump 500 operates to pumpeither sample fluid or buffer fluid into MAG 123. A flow limiter 509/510may be attached to the output line 201 from pump 500 to reduce flow ratepulsation from the pump 500. Channel line 201 is mounted onto MAG 123.Output line 925 connects to MAG output sample container 934. Output line926 connects to MAG output sample container 9342. Valve 940 is attachedto line 925 and valve 937 is attached to line 926, which controlnegative entities and positive entities from MAG 123 going into eithercontainer 934 or container 9342. Valves 940 and 937 may both shut downthe flow in lines 925 and 926 during demagnetization/dissociationprocess of MAG 123. FIG. 59B shows containers 928, 929, 934 and 9342 maybe in the form of blood bags, but they may also be in other physicalforms of vial or bottles.

FIG. 60A illustrates embodiment of closed and disposable fluidic linesfor sample processing through a single UFL 600. Input line 9271 mayconnect to a UFL sample liquid container, and also connects to inlet ofa first pump tubing 504/505, which further connects to entity input line6020 of UFL 600. Input line 9272 may connect to a UFL buffer container,and also connects to inlet of a second pump tubing 504/505, whichfurther connects to buffer input line 6040 of UFL 600. UFL 600 largeentity output line 6070 may connect to a large entity container. UFL 600small entity output line 6090 may connect to a small entity container.

FIG. 60B illustrates fluidic lines of FIG. 60A being connected to, orattached with, various fluidic components. First and second pump tubings504/505 are each installed into a peristaltic pump 500. The two pumps500 operate to pump sample fluid and buffer fluid into UFL 600. A flowlimiter 509/510 may be attached to the output line from each pump 500,including lines 6020 and 6040, to reduce flow rate pulsation from thepump 500. Input line 9271 connects to a liquid sample container 928.Input line 9272 connects to UFL buffer container 931. UFL output line6070 connects to large entity container 932. UFL output line 6090connects to small entity container 933. Adjustable valves 939 and 938may be attached to the lines 6070 and 6090 to adjust the flow ratewithin each of lines 6070 and 6090, which in turn controls the laminarflow speed in UFL 600 channel for channel center buffer flow and channeledge entity sample flow. FIG. 60B shows containers 928, 931, 932 and 933may be in the form of blood bags, but they may also be in other physicalforms of vial or bottles without limitation.

FIG. 61A illustrates replacement of peristaltic pumps of FIG. 56B withpressurized chambers 800 on input sample bags to drive fluid throughfluidic lines. In FIG. 61A, pumps 500, pump tubings 504/505, and flowlimiters 509/510 of FIG. 56B are removed. Channel 201 is connecteddirectly to T-connector 921. Connector 801 is replaced with connector803 bag. UFL entity liquid line 6020 is connected to connector 803.Sample liquid bag 928, MAG buffer bag 929, connector 803 bag and UFLbuffer bag 931 are each enclosed in a pressure chamber 800. Pressurechamber 800 may operate by increasing pressure of chamber medium, forexample air or other fluid, where the bags enclosed in chambers aresubmerged in the chamber medium. With increase in chamber mediumpressure, liquid contained in the bags may be forced out of the bags andinto the fluid lines. FIG. 61A operation may need separate MAG 123 andUFL 600 operations. At first stage, valve 941 attached to line 6020closes. Pressure in chambers 800 enclosing bags 803 and 931 is released.Pressures in chambers 800 enclosing bags 928 and 929 are increased toforce sample fluid or buffer fluid into channel 201 to start MAG 123separation. After MAG 123 separation and sample fluid in bag 928 isdepleted, bag 934 and connector 803 are each filled with output samplesfrom MAG 123 after MAG separation. Then, at second stage, valve 937 isclosed and valve 941 is opened. Chambers 800 around connector 803 andbag 931 increase pressure to force connector 803 sample and buffer fluidin 931 to flow into the UFL 600 to start UFL separation. After sample inconnector 803 is depleted, and UFL 600 separation is finished, bags 932and 933 contain large and small entities from UFL output. Connector 803maybe replaced by connector 8020 of FIG. 52 which has an air port 8023.

FIG. 61B illustrates replacement of peristaltic pumps of FIG. 56B withvacuum chambers 806 on output sample bags to drive fluid through fluidiclines. FIG. 61B is same as FIG. 61A, except pressure chambers 800 areremoved. Bag 934, 932, 933, and connector 803 are each enclosed in avacuum chamber 806. Vacuum chamber 806 may operate by increasing vacuumlevel within each chamber 806, where fluid from the fluid linesconnected to the bags is forced into the bags enclosed in chambers dueto fluid line pressure being larger than the vacuum pressure. FIG. 61Boperation may also need separate MAG 123 and UFL 600 operations. Atfirst stage, valve 941 attached to line 6020 closes. Vacuum in chambers806 enclosing bags 932 and 933 is released. Vacuum in chambers 806enclosing bags 934 and 803 are increased to force sample fluid or bufferfluid into channel 201 to start MAG 123 separation. After MAG 123separation and sample fluid in bag 928 is depleted, bag 934 andconnector 803 are each filled with output samples from MAG 123 after MAGseparation. Then, at second stage, valve 937 is closed and valve 941 isopened. Vacuum in chamber 806 around connector 803 is released. Vacuumsin chambers 806 enclosing bags 932 and 933 are increased to forceconnector 803 sample and buffer fluid in container 931 to flow into theUFL 600 to start UFL separation. After sample in connector 803 isdepleted, and UFL 600 separation is finished, bags 932 and 933 containlarge and small entities from UFL output. Connector 803 maybe replacedby connector 8020 of FIG. 52 which has an air port 8023.

FIG. 62A illustrates replacement of peristaltic pumps of FIG. 57B withpressurized chambers 800 on input sample bags to drive fluid throughfluidic lines. In FIG. 62A, pumps 500, pump tubings 504/505, and flowlimiters 509/510 of FIG. 57B are removed. Channel 201 is connecteddirectly to T-connector 921. Connector 801 is replaced with connector803 bag. MAG sample line 923 is connected to connector 803. Sampleliquid bag 928, MAG buffer bag 929, connector 803 bag and UFL buffer bag931 are each enclosed in a pressure chamber 800. FIG. 62A may separateUFL 600 and MAG 123 operations. At first stage, valve 935 attached toline 923 closes. Pressure in chamber 800 enclosing bag 803 is released.Pressures in chambers 800 enclosing bags 928 and 931 are increased toforce sample fluid and UFL buffer fluid into UFL 600 inlets to start UFL600 separation. After UFL 600 separation and sample fluid in bag 928 isdepleted, bag 933 contains small entity fluid and connector 803 containslarge entity fluid from UFL 600 separation. Then, at second stage, valve939 is closed and valve 935 is opened. Chambers 800 around connector 803and bag 929 increase in pressure to force connector 803 large entityfluid sample or MAG buffer fluid in bag 929 to flow into channel 201 ofMAG 123 to start MAG 123 separation. After sample in connector 803 isdepleted, and MAG 123 separation is finished, bags 934 and 9342 containpositive sample and negative sample from MAG 123 channel 201 output.Connector 803 maybe replaced by connector 8020 of FIG. 52 .

FIG. 62B illustrates replacement of peristaltic pumps of FIG. 57B withvacuum chambers 806 on output sample bags to drive fluid through fluidiclines. FIG. 62B is same as FIG. 62A, except pressure chambers 800 areremoved. Bag 934, 9342, 933, and connector 803 are each enclosed in avacuum chamber 806. FIG. 62B operation may separate MAG 123 and UFL 600operations. At first stage, valve 935 attached to line 923 closes.Vacuum in chambers 806 enclosing bags 933 and 803 are increased to forcesample fluid and UFL buffer fluid into inlets of UFL 600 to start UFL600 separation. After UFL 600 separation and sample fluid in bag 928 isdepleted, bag 933 contains small entity fluid and connector 803 containslarge entity fluid from UFL 600 separation. Then, at second stage,valves 938 and 939 are closed and valve 923 is opened. Vacuum in chamber806 around connector 803 is released. Vacuums in chambers 806 enclosingbags 934 and 9342 are increased to force connector 803 large entitysample or MAG buffer fluid in bag 929 to flow into channel 201 of MAG123 to start MAG 123 separation. After sample in connector 803 isdepleted, and MAG 123 separation is finished, bags 934 and 9342 containpositive sample and negative sample from MAG 123 channel 201 output.Connector 803 maybe replaced by connector 8020 of FIG. 52 .

FIG. 63A illustrates replacement of peristaltic pumps of FIG. 58B withpressurized chambers 800 on input sample bags to drive fluid throughfluidic lines. FIG. 63A is identical to FIG. 62A in fluid line layoutand in operation of UFL 600 and MAG 123 with chambers 800, except thatthe UFL large entity output 6070 connects to large entity container 932in blood bag form, and small entity output 6090 connects to connector803.

FIG. 63B illustrates replacement of peristaltic pumps of FIG. 58B withvacuum chambers 806 on output sample bags to drive fluid through fluidiclines. FIG. 63B is identical to FIG. 62B in fluid line layout and inoperation of UFL 600 and MAG 123 with chambers 806, except that the UFLlarge entity output 6070 connects to large entity container 932 in bloodbag form with large entity container 932 enclosed in vacuum chamber 806replacing container 933 of FIG. 62B, and small entity output 6090connects to connector 803.

FIG. 64A illustrates replacement of peristaltic pumps of FIG. 59B withpressurized chambers 800 on input sample bags 928 and 929 to drive fluidthrough channel 201 of MAG 123. In FIG. 64A, pump 500, pump tubing504/505, and flow limiter 509/510 of FIG. 59B are removed. Channel 201is connected directly to T-connector 921. Sample liquid bag 928 and MAGbuffer bag 929 are each enclosed in a pressure chamber 800. Pressures inchambers 800 enclosing bags 928 and 929 are increased to force samplefluid or buffer fluid into channel 201 to start MAG 123 separation.After MAG 123 separation and sample fluid in bag 928 is depleted, bag934 and bag 9342 are each filled with either negative entities orpositive entities from MAG 123 after MAG separation.

FIG. 64B illustrates replacement of peristaltic pumps of FIG. 59B withvacuum chambers 806 on output sample bags 934 and 9342 to drive fluidthrough channel 201 of MAG 123. FIG. 64B is same as FIG. 64A, exceptpressure chambers 800 are removed. Output sample bags 934 and 9342 areeach enclosed in a vacuum chamber 806. Vacuums in chambers 806 enclosingbags 934 and 9342 are increased to force entity sample from bag 928 orMAG buffer fluid from bag 929 to flow into channel 201 of MAG 123 tostart MAG 123 separation. After sample in bag 928 is depleted, and MAG123 separation is finished, bags 934 and 9342 contain positive sampleand negative sample from MAG 123 channel 201 output.

FIG. 65A illustrates replacement of peristaltic pumps of FIG. 60B withpressurized chambers 800 on sample liquid bag 928 and UFL buffer bag 931to drive fluid through UFL 600. In FIG. 65A, pump 500, pump tubing504/505, and flow limiter 509/510 of FIG. 60B are removed. Sample liquidbag 928 and UFL buffer bag 931 are each enclosed in a pressure chamber800. Pressures in chambers 800 enclosing bags 928 and 931 are increasedto force sample fluid and UFL buffer fluid into UFL 600 inlets to startUFL 600 separation. After UFL 600 separation, sample fluid in bag 928 isdepleted, bag 932 contains large entity fluid and bag 933 contains smallentity fluid.

FIG. 65B illustrates replacement of peristaltic pumps of FIG. 60B withvacuum chambers 906 on output sample bags 932 and 933 to drive fluidthrough UFL 600. FIG. 65B is same as FIG. 65A, except pressure chambers800 are removed. Output sample bags 932 and 933 are each enclosed in avacuum chamber 806. Vacuums in chambers 806 enclosing bags 932 and 933are increased to force sample liquid from bag 928 and UFL buffer fluidfrom bag 931 to flow through UFL 600 to start UFL separation. Aftersample in bag 928 is depleted, and UFL 600 separation is finished, bag932 contains large entity fluid and bag 933 contains small entity fluid.

Structures, components, and methods as described from FIG. 55A throughFIG. 65B on enclosed fluidic lines including one UFL 600 and one MAG123, may be applied to FIG. 47 through FIG. 52 without limitation, whereenclosed fluidic lines including multiple MAGs 123 and multiple UFLs 600may be achieved by replicating the components on single UFL 600 andsingle MAG 123 from FIG. 55A through FIG. 65B on each of the UFLs 600and MAGs 123 of FIG. 47 through FIG. 52 .

FIG. 66 through FIG. 88 illustrate embodiments of process flows toutilize MAG and UFL devices to separate biological entities from variousbiological samples. For simplicity of description, terms UFL and MAG areused in these figures for explanation. However, UFL may be any of UFL600, 650, 620, 630, 640 of FIG. 40A, FIG. 41A, FIG. 42A, FIG. 43 , whileMAG may be any of MAG 121, 122, 123, 124, 124,125, 126, 127, 128, 129with corresponding channel types as described in prior figures withoutlimitation and without sacrifice of performance. If a component, or astructure, in FIG. 66 through FIG. 88 shares same name with anothercomponent or structure in prior figures, it then means the samecomponent, or same structure as that in prior figures.

FIG. 66 illustrates embodiment of a first process flow to separatebiological entities from peripheral blood using UFL and MAG. In step5801, peripheral blood sample is collected from a patient or personunder test. In step 5802, red blood cell lysing may be performed on saidperipheral blood sample, where step 5802 in another embodiment may beskipped. In step 5803, said blood sample from step 5802, or directlyfrom step 5801, is injected into UFL entity fluid inlet 602, while UFLbuffer fluid is injected into outlet 604. In step 5804, frequency andvibration strength of PZT attached to UFL are set to produce a standingwave and pressure nodes in UFL fluid. In step 5805, UFL outlet 607outputs target sample that contains large size entities or cells. Instep 5806, magnetic labels hybridized with antibodies or ligands, whichspecifically bind to surface antigens or receptors on target cells orentities, are added into target sample from step 5805. In step 5807,target sample from step 5806 is incubated to form magnetic labelsbinding to target cells or entities. In step 5808, target sample fromstep 5807 is flowed through MAG channel at magnetic separation positon,where during step 5808, negative MAG sample may be forwarded as in step5815 to be collected in step 5813. In step 5809, target cells orentities bound with magnetic label are separated by MAG within the MAGchannel; in step 5810, after step 5809, buffer fluid may be flowedthrough MAG channel to wash out residual non-target entities withoutmagnetic labels. The washed out fluid may be forwarded as in step 5816to be collected as negative MAG sample in step 5813. Step 5810 may beskipped in another embodiment. In step 5811, after step 5810 or directlyafter step 5809, separated entity conglomerate in MAG channel may bedissociated into isolated cells or entities. In step 5812, buffer fluidis flowed through MAG channel to wash out dissociated cells and entitiesin MAG channel, which, as shown by path 5817, may be collected aspositive MAG sample in step 5814.

Peripheral blood sample of FIG. 66 may also be other body fluids,including but not limited to: saliva, tear, mucus, urine, secretion fromvarious organs of body.

FIG. 67 illustrates an embodiment of second process flow to separatebiological entities from peripheral blood using MAG. Every other aspectof FIG. 67 is same as FIG. 66 , except step 5803, step 5804, and step5805 of FIG. 66 are removed between step 5802 and step 5806 in FIG. 67 .While in FIG. 67 , blood sample from step 5802, or blood sample directlyfrom step 5801, is centrifuged in step 6201 to extract target samplecontaining white blood cells. Target sample form step 6201 is then sentto step 5806. From step 5806, process flow in FIG. 67 is same as that inFIG. 66 .

FIG. 68 illustrates an embodiment of third process flow to separatebiological entities from peripheral blood using MAG. Every other aspectof FIG. 68 is same as FIG. 66 , except step 5803, step 5804, and step5805 of FIG. 66 are removed between step 5802 and step 5806 in FIG. 68 .In FIG. 68 , peripheral blood sample collected from patient or personunder test in step 6301, which is same as step 5801 of FIG. 66 , isregarded as target sample. Target sample from step 5802 after red bloodcell lysing, which is after step 6301, or directly from step 6301, isthen sent to step 5806. From step 5806, process flow in FIG. 68 is sameas that in FIG. 66 .

FIG. 69 illustrates an embodiment of fourth process flow to separatebiological entities from peripheral blood using MAG. Every other aspectof FIG. 69 is same as FIG. 66 , except step 5801, step 5802, step 5803,step 5804, and step 5805 of FIG. 66 are removed before step 5806 in FIG.69 . In FIG. 69 , target sample is collected after apheresis ofperipheral blood sample collected from patient or person under test.Target sample from step 6401 is then sent to step 5806. From step 5806,process flow in FIG. 69 is same as that in FIG. 66 .

FIG. 70 illustrates an embodiment of fifth process flow to separatebiological entities from tissue sample using UFL and MAG. Every otheraspect of FIG. 70 is same as FIG. 66 , except step 5801, step 5802, andstep 5803 are removed before step 5804 in FIG. 70 . In FIG. 70 , tissuesample is collected in step 6501. In step 6502, tissue sample from step6501 is dissociated in a fluid base. In step 6503, dissociated tissuefluid of step 6502 is injected into UFL channel through inlet 602 andUFL buffer fluid is injected through inlet 604. From step 5804, processflow in FIG. 70 is same as that in FIG. 66 . Tissue sample of FIG. 70may include any of: human body tissue aspirate, human organ tissueaspirate, bone marrow, animal body or organ tissue aspirate. Targetcells or entities of FIG. 70 may be rare disease cells, for examplecancer cells, or micro-organisms, for example bacteria.

FIG. 71 illustrates an embodiment of sixth process flow to separatebiological entities from tissue sample using MAG. Every other aspect ofFIG. 71 is same as FIG. 70 , except step 6503, step 5804, and step 5805are removed before step 5806 in FIG. 71 . In FIG. 71 , tissue samplefrom step 6501 is dissociated in a fluid base in step 6502 to formtarget sample, and process continues to step 5806. From step 5806,process flow in FIG. 71 is same as that in FIG. 70 .

FIG. 72 illustrates an embodiment of seventh process flow to separatebiological entities from surface swab sample using UFL and MAG. Everyother aspect of FIG. 72 is same as FIG. 66 , except step 5801, step5802, and step 5803 are removed before step 5804 in FIG. 72 . In FIG. 72, surface entities are collected in step 6701 by swab. In step 6702,surface entities collected on swab are dissolved in a fluid base. Instep 6703, fluid base with dissolved surface entities from step 6702 isinjected into UFL channel through inlet 602 and UFL buffer fluid isinjected through inlet 604. From step 5804, process flow in FIG. 72 issame as that in FIG. 66 . Surface entities of FIG. 72 may be collectedby swab from subjects including any of: human body, saliva, body fluid,human body discharge, animal, plant, soil, air, water, and merchandise.Target cells or entities of FIG. 72 may include cells from human body,or animal body, or plant, or include micro-organisms, for examplebacteria, mold, or spores.

FIG. 73 illustrates an embodiment of eighth process flow to separatebiological entities from surface swab sample using MAG. Every otheraspect of FIG. 73 is same as FIG. 72 , except step 6703, step 5804, andstep 5805 are removed before step 5806 in FIG. 73 . In FIG. 73 , surfaceentities collected on swab in step 6701 are dissolved in a fluid base instep 6702 to form target sample, and process continues to step 5806.From step 5806, process flow in FIG. 73 is same as that in FIG. 72 .

FIG. 74 illustrates an embodiment of ninth process flow to separatebiological entities from solid sample using UFL and MAG. Every otheraspect of FIG. 74 is same as FIG. 66 , except step 5801, step 5802, andstep 5803 are removed before step 5804 in FIG. 74 . In FIG. 74 , solidsample is collected in step 6901. In step 6902, solid sample from step6901 is dissociated in a fluid base. In step 6903, dissociated solidsample fluid of step 6902 is injected into UFL channel through inlet 602and UFL buffer fluid is injected through inlet 604. From step 5804,process flow in FIG. 74 is same as that in FIG. 66 . Solid sample ofFIG. 74 may include any of: solid biological products or waste generatedby human, animal, or plant, powder, and soil. Target cells or entitiesof FIG. 74 may include cells from human body, or animal body, or plant,or include micro-organisms, for example bacteria, mold, or spores.

FIG. 75 illustrates an embodiment of tenth process flow to separatebiological entities from solid sample using MAG. Every other aspect ofFIG. 75 is same as FIG. 74 , except step 6903, step 5804, and step 5805are removed before step 5806 in FIG. 75 . In FIG. 75 , solid sample fromstep 6901 is dissociated in a fluid base in step 6902 to form targetsample, and target sample is continuously processed in step 5806. Fromstep 5806, process flow in FIG. 75 is same as that in FIG. 74 .

FIG. 76A illustrates addition of both magnetic and fluorescent labelsinto fluid samples for specific binding to target cells or entities.FIG. 76A shows that step 5806 of FIG. 66 through FIG. 75 may be modifiedto become step 58061, where in addition to magnetic labels, fluorescentlabels hybridized with antibodies or ligands, which specifically bind tosurface antigens or receptors on target cells or entities, may also beadded in target sample from step 5805.

FIG. 76B then illustrates that incubation step 5807 of FIG. 66 throughFIG. 75 may also be modified to become step 58071, which includesincubation of both magnetic and fluorescent labels at the same time toform specific binding to target cells or entities. Binding sites ofmagnetic labels and fluorescent labels on same target cells or entitiesmay be different.

Steps 5806 and step 58061 may be realized in a flow connector includingany one of 801, 802, 803, 8010, 8020, 8030 of prior figures, where flowconnector may contain pre-filled hybridized magnetic labels andfluorescent labels in liquid solution, or in dry powder form. Step 5807and step 58071 may also occur in said flow connector, where said flowconnector may also be located in a temperature control chamber tocontrol incubation speed and quality. In another embodiment, said flowconnector may have attached or embedded temperature control circuit tocontrol incubation in flow connector.

FIG. 77A illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL before magnetic separation by MAG. FIG. 77Ashows that for each of FIG. 66 through FIG. 75 , step 5818 and step 5819may be added between step 5807 and step 5808. After target sample isincubated in step 5807, in step 5818, target sample may be injected intosecond UFL through inlet 602, and buffer fluid may be injected intosecond UFL through inlet 604. In step 5819, second UFL outputs targetsample containing large entities from outlet 607, and non-bound freemagnetic labels are output from second UFL outlet 609. Then in step5808, target sample containing large entities from second UFL outlet 607is passed through MAG channel for magnetic separation. Target sample instep 5819 may contain cells 10/30 or entities bound with magneticlabels. Second UFL having an attached PZT that operates with a specifiedultrasound vibration amplitude and frequency to create a standing wavein second UFL channel fluid is assumed in step 5819.

FIG. 77B illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL after magnetic separation by MAG. FIG. 77Bshows that for each of FIG. 66 through FIG. 75 , step 5820 and step 5821may be added between step 5812 and step 5814, replacing path 5817. Aftermagnetic conglomerate within MAG channel is dissociated and the positiveMAG sample entities from MAG channel are flushed out as in step 5812,flushed out positive MAG sample may be injected into third UFL throughinlet 602, and buffer fluid may be injected into third UFL through inlet604. In step 5821, third UFL outputs positive MAG sample containinglarge entities from outlet 607, and non-bound free magnetic labels areoutput from third UFL outlet 609. Then in step 5814, positive MAG samplewith reduced or depleted free magnetic labels may be collected. ThirdUFL having an attached PZT that operates with a specified ultrasoundvibration amplitude and frequency to create a standing wave in third UFLchannel fluid is assumed in step 5821.

FIG. 78A illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL before magneticseparation by MAG. FIG. 78A is similar to FIG. 77A, with step 5807 ofFIG. 77A being replaced by step 58071 of FIG. 76B, and step 5819 beingreplaced by step 58191. After adding magnetic labels and fluorescentlabels into target sample as in step 58061 of FIG. 76A, target sample isincubated in step 58071, same as in FIG. 76B, to form magnetic label andfluorescent label binding to target cells or entities. In step 5818,target sample may be injected into second UFL through inlet 602, andbuffer fluid may be injected into second UFL through inlet 604. In step58191, second UFL outputs target sample containing large entities fromoutlet 607, and non-bound free magnetic labels and free fluorescentlabels are output from second UFL outlet 609. Then in step 5808, targetsample containing large entities from second UFL outlet 607 is flownthrough MAG channel for magnetic separation. Target sample in step 58191may contain cells 30 or entities bound with magnetic and fluorescentlabels. Second UFL having an attached PZT that operates with a specifiedultrasound vibration amplitude and frequency to create a standing wavein second UFL channel fluid is assumed in step 58191.

FIG. 78B illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL after magneticseparation by MAG. FIG. 78B is similar to FIG. 77B, with step 5821 ofFIG. 77A being replaced by step 58211. Separated entities in step 5812and step 5820 of FIG. 78B may contain: cells 30 or entities bound withmagnetic and fluorescent labels, non-bound free magnetic labels, andsmall amount of non-bound free fluorescent labels due to non-specificbinding to conglomerate in MAG channel during magnetic separation. Instep 58212, third UFL outputs positive MAG sample containing largeentities from outlet 607, and non-bound free magnetic and free opticallabels are output from third UFL outlet 609. Then in step 5814, positiveMAG sample with reduced or depleted free magnetic labels and freefluorescent labels may be collected. Third UFL having an attached PZTthat operates with a specified ultrasound vibration amplitude andfrequency to create a standing wave in third UFL channel fluid isassumed in step 58212.

FIG. 79 illustrates continued process of negative MAG sample after MAGseparation, as in step 408 of FIG. 31 , through UFL to remove smallentities and passing of large entities into various cell processingdevices and procedures. Step 5813 is same as that in FIG. 66 throughFIG. 75 , where negative MAG sample is collected during MAG separationof a target sample. In step 5822, negative MAG sample of step 5813 isinjected into fourth UFL inlet 602, and UFL buffer is injected intoinlet 604 of fourth UFL. In step 5823, fourth UFL outputs negative MAGsample containing large entities from outlet 607, and small sizeentities are removed from large entities and output from fourth UFLoutlet 609, and a PZT that attaches to fourth UFL and operates with aspecified ultrasound vibration amplitude and frequency to create astanding wave in fourth UFL is assumed to be used. Finally, negative MAGsample containing large entities from outlet 607 of fourth UFL may besent to be analyzed by any of: cell counter 903, cell imager 904, flowcytometer or sorter 905, DNA/RNA sequencer 906. Alternatively, outputfrom cell counter 903, or output from cell imager 904, or output fromflow cytometer or sorter 905, may be further sent to be processed byDNA/RNA sequencer 906 as indicated respectively by paths 936, 946, and956. Negative MAG sample containing large entities from outlet 607 offourth UFL in step 5823 may also be sent into the process of cellgenetic modification and cell expansion 5824. Prior to DNA/RNAsequencing in DNA/RNA sequencer 906, a polymerase chain reaction (PCR)procedure on DNA/RNA sample obtained from cell lysing of large sizeentities from outlet 607 of fourth UFL from step 5823 may be performed,where PCR may be targeting one or more target DNA/RNA sequences andamplifies the number of target DNA/RNA sequences in the DNA/RNA sample.

FIG. 80 illustrates continued process of negative MAG sample after MAGseparation, as in step 408 of FIG. 31 , through UFL to retrieve smallentities and passing of small entities into various molecule or smallentity processing devices. After step 5813 of FIG. 66 through FIG. 75 ,where negative MAG sample is collected during MAG separation of a targetsample, in step 5822, negative MAG sample of step 5813 is injected intofourth UFL inlet 602, and UFL buffer is injected into inlet 604 offourth UFL. In step 5825, fourth UFL outputs negative MAG samplecontaining large entities from outlet 607, and small size entitiesincluding DNA, RNA, molecules, and other small particles are output fromfourth UFL outlet 609, and a PZT that attaches to fourth UFL andoperates with a specified ultrasound vibration amplitude and frequencyto create a standing wave in fourth UFL is assumed to be used. Finally,small size entities from outlet 609 of fourth UFL may be sent to beanalyzed by any of: particle counter 5835, particle imager 5836, flowcytometer or sorter 905, DNA/RNA sequencer 906. Alternatively, outputfrom particle counter 5835, or output from particle imager 5836, oroutput from flow cytometer or sorter 905, may be further sent to beprocessed by DNA/RNA sequencer 906 as indicated respectively by paths5827, 5828, and 956. DNA/RNA sequencer 906 may contain a PCR step onsmall size entities from outlet 609 of fourth UFL from step 5825 priorto DNA/RNA sequencing, where PCR may target one or more particularDNA/RNA sequences to amplify their quantity.

FIG. 81 illustrates entity analysis of negative MAG sample after MAGseparation, as in step 407 of FIG. 31 , using various analyzing devices.After step 5813 of FIG. 66 through FIG. 75 , where negative MAG sampleis collected during MAG separation of a target sample, collectednegative MAG sample may be sent to be analyzed by any of: cell counter903, cell imager 904, flow cytometer or sorter 905, particle counter5835, particle imager 5836, DNA/RNA sequencer 906. Alternatively, outputfrom cell counter 903, or output from cell imager 904, or output fromflow cytometer or sorter 905, or output from particle counter 5835, oroutput from particle imager 5836, may be further sent to be processed byDNA/RNA sequencer 906 as indicated respectively by paths 936, 946, 956,5827, and 5828. Negative MAG sample may also be sent into the process ofcell genetic modification and cell expansion 5824. DNA/RNA sequencer 906may contain a PCR step on: (1) DNA/RNA obtained after cell lysing ofcells contained within negative MAG sample; and (2) DNA/RNA/moleculescontained within negative MAG sample. Prior to DNA/RNA sequencing, PCRmay target one or more particular DNA/RNA sequences to amplify theirquantity.

FIG. 82 illustrates continued process of positive MAG sample after MAGseparation, as in step 408 of FIG. 31 , through UFL to remove smallentities and passing of large entities into various cell processingdevices and procedures. Step 5814 is same as that in FIG. 66 throughFIG. 75 , where positive MAG sample is collected after MAG separation ofa target sample. In step 5829, positive MAG sample of step 5814 isinjected into fifth UFL inlet 602, and UFL buffer is injected into inlet604 of fifth UFL. In step 5830, fifth UFL outputs positive MAG samplecontaining large entities from outlet 607, and small size entities areremoved from large entities and output from fifth UFL outlet 609, and aPZT that attaches to fourth UFL and operates with a specified ultrasoundvibration amplitude and frequency to create a standing wave in fifth UFLis assumed to be used. Finally, positive MAG sample containing largeentities from outlet 607 of fifth UFL may be sent to be analyzed by anyof: cell counter 903, cell imager 904, flow cytometer or sorter 905,DNA/RNA sequencer 906. Alternatively, output from cell counter 903, oroutput from cell imager 904, or output from flow cytometer or sorter905, may be further sent to be processed by DNA/RNA sequencer 906 asindicated respectively by paths 936, 946, and 956. Positive MAG samplecontaining large entities from outlet 607 of fifth UFL in step 5830 mayalso be sent into the process of cell genetic modification and cellexpansion 5824. DNA/RNA sequencer 906 may contain a PCR step on DNA/RNAobtained after cell lysing of large size entities from outlet 607 offifth UFL from step 5830 prior to DNA/RNA sequencing, where PCR maytarget one or more particular DNA/RNA sequences to amplify theirquantity.

FIG. 83 illustrates continued process of positive MAG sample after MAGseparation, as in step 408 of FIG. 31 , through UFL to retrieve smallentities and passing of small entities into various molecule or smallentity processing devices. After step 5814 of FIG. 66 through FIG. 75 ,where positive MAG sample is collected after MAG separation of a targetsample, in step 5829, positive MAG sample of step 5814 is injected intofifth UFL inlet 602, and UFL buffer is injected into inlet 604 of fifthUFL. In step 5831, fifth UFL outputs positive MAG sample containinglarge entities from outlet 607, and small size entities including DNA,RNA, molecules, and other small particles bound by magnetic labels areoutput from fifth UFL outlet 609, and a PZT that attaches to fifth UFLand operates with a specified ultrasound vibration amplitude andfrequency to create a standing wave in fifth UFL is assumed to be used.Finally, small size entities from outlet 609 of fifth UFL may be sent toany of: particle counter 5835, particle imager 5836, flow cytometer orsorter 905, DNA/RNA sequencer 906. Alternatively, output from particlecounter 5835, or output from particle imager 5836, or output from flowcytometer or sorter 905, may be further sent to be processed by DNA/RNAsequencer 906 as indicated respectively by paths 5827, 5828, and 956.DNA/RNA sequencer 906 may contain a PCR step on small size entities fromoutlet 609 of fifth UFL from step 5831 prior to DNA/RNA sequencing,where PCR may target one or more particular DNA/RNA sequences to amplifytheir quantity.

FIG. 84 illustrates entity analysis of positive MAG sample after MAGseparation, as in step 407 of FIG. 31 , using various analyzing devices.After step 5814 of FIG. 66 through FIG. 75 , where positive MAG sampleis collected after MAG separation of a target sample, collected positiveMAG sample may be sent to be analyzed by any of: cell counter 903, cellimager 904, flow cytometer or sorter 905, particle counter 5835,particle imager 5836, DNA/RNA sequencer 906. Alternatively, output fromcell counter 903, or output from cell imager 904, or output from flowcytometer or sorter 905, or output from particle counter 5835, or outputfrom particle imager 5836, may be further sent to be processed byDNA/RNA sequencer 906 as indicated respectively by paths 936, 946, 956,5827, and 5828. Positive MAG sample may also be sent into the process ofcell genetic modification and cell expansion 5824. DNA/RNA sequencer 906may contain a PCR step on: (1) DNA/RNA obtained after cell lysing ofcells contained within positive MAG sample; and (2) DNA/RNA/moleculescontained within positive MAG sample, prior to DNA/RNA sequencing, wherePCR may target one or more particular DNA/RNA sequences to amplify theirquantity.

FIG. 85A illustrates addition of fluorescent labels to specifically bindto target entities within negative MAG sample immediately after negativeMAG sample collection. FIG. 85A shows that in step 58131, immediatelyafter step 5813, where negative MAG sample is collected during MAGseparation, fluorescent labels, which are hybridized with antibodies orligands and specifically bind to surface antigens or receptors on targetcells or entities, are added into the negative MAG sample, and thennegative MAG sample is incubated to form fluorescent labels bound totarget cells or entities. Step 58131 may be inserted between step 5813and step 5822 in FIG. 79 and FIG. 80 , or inserted immediately afterstep 5813 and before devices or processes 903, 904, 905, 906, 5824,5825, and 5826 in FIG. 81 .

FIG. 85B illustrates addition of fluorescent labels to specifically bindto target entities within positive MAG sample immediately after positiveMAG sample collection. FIG. 85B shows that in step 58141, immediatelyafter step 5814, where positive MAG sample is collected after MAGseparation, fluorescent labels, which are hybridized with antibodies orligands and specifically bind to surface antigens or receptors on targetcells or entities, are added into the positive MAG sample, and thenpositive MAG sample is incubated to form fluorescent labels bound totarget cells or entities. Step 58141 may be inserted between step 5814and step 5829 in FIG. 82 and FIG. 83 , or inserted immediately afterstep 5814 and before devices or processes 903, 904, 905, 906, 5824,5825, and 5826 in FIG. 84 .

FIG. 86A through FIG. 93 describe methods to achieve pre-symptom earlystage tumor detection, especially in asymptomatic tumor patients who arein very early stage, or have not been diagnosed with tumor, or areshowing no symptom, or have tumor that is in such infancy or early stagethat may not be detected or located by conventional methods, includingimaging or blood test. In the description, terms of “cancer” and “tumor”may be used interchangeably and have same meaning.

Malignant tumor, or cancer, is a disease that results from geneticmutation of normal body cells, which become astray from original cellfunctions, multiply fast, and evade normal cell life cycle of programmedcell death by human immune system. To increase the survival chance of apatient carrying cancer, it is imperative to identify and locate thecancer at the earliest stage possible. In state-of-art medicinepracticed today, tumors are still found or identified either afterphysical identification by imaging methods including ultrasound, X-ray,computerized tomography (CT), magnetic resonance imaging (MRI), or inmost cases after patient showing symptoms due to cancer growth. Forcancer to be identified by imaging methods or by patient showingsymptoms, cancer growth is typically already underway, and in most caseswell developed with cancer cells in the body already growing insignificant numbers. For certain cancers, for example pancreatic cancerwhich is typically asymptomatic even in late stages, detection byconventional method would usually be too late to provide meaningfulmedical intervention. It is desirable, and imperative, to have a cancerdetection method that is able to detect occurrence of a cancer, withlocation of origination, at its infancy stage, where such detection ispreferred before cancer's significant growth, and before any statisticalpossibility of cancer's spreading from a local growth to other parts ofbody. This detection method is desirable to be administered to aperson-under-test through conventional clinical means, for exampletypical peripheral blood collection and blood test. By achievingpre-symptom early stage cancer detection and knowing with confidence ofcancer type and location, medical intervention may provide mosteffectiveness in removal of cancer cells, significantly increasesurvival rate, and eventually cure cancer, and at the same timesignificantly reduces financial and social burden of cancer treatment.

FIG. 86A illustrates first example of cancer treatment. Cancer cell 2002exhibits surface antigen, ligand or surface marker 2004, which can beused to identity and kill cancer cell 2002 in human body by an immunecell 2001 with a surface anti-body, or receptor 2003 that specificallybinds to surface marker 2004, as shown by bond 2034. Immune cell 2001may be extracted from the person-under-test (PUT), or from a donorperson. Immune cell 2001 may be genetically modified after beingcollected from the PUT or donor to express surface receptor 2003. Immunecell 2001 with antibody 2003 may be expanded or cultivated in ex vivoenvironment. Immune cell 2001 may be engineered to suppress, or evade,immune system response of the PUT if immune cell is collected fromdonor. In practice, immune cells 2001 with antibody 2003 may beadministered to PUT with blood infusion, where immune cells 2001 willthen find and bind to cancer cells 2002 in vivo through antibody-antigenbond 2034 between antibody 2003 and marker 004 and kill the cancer cells2002. For example, cell 2001 may be a type of ex vivo engineeredchimeric antigen receptor T cell (CAR-T) with receptor 2003 beingchimeric antigen receptor (CAR) that targets cancer cell 2002 which hasa surface marker 2004. Receptor 2003 may be any of, but not limited to,CD19, CD20, CD22, CD30, ROR1, light chain, CD123, CD33, CD133, CD138,and B-cell maturation antigen.

FIG. 86B illustrates second example of cancer treatment. In PUT, theperson's internal immune cell 2007, for example a T cell, functions toidentify cells which need to be terminated as a normal cell life cycle.For a normal cell, immune cell 2007 forms receptor 2051 to antigen 2052bond, which enables immune cell 2007 to terminate a normal cell at endof life cycle of the normal cell. Receptor 2501 may include T-cellreceptor (TCR), CD 28. However, cancer cell 2002 evades such programmeddeath from immune cell 2007 by forming another ligand 2054 to receptor2053 bond with immune cell 2007, which effectively disables the receptor2051 to antigen 2052 bond that functions to terminate the cancer cell2002. For example, ligand 2054 and receptor 2053 may be PD-L1 and PD-1,respectively. In FIG. 86B method, antibody 2056 or antigen 2055 may beadministered to PUT, such that antibody 2056 may bind to ligand 2054 andantigen 2055 may bind to receptor 2053 in vivo of PUT body, causingeffective disconnection of receptor 2053 to ligand 2054 bond, and makingtermination of the cancer cell 2002 by the immune cell 2007 possiblethrough receptor 2051 to antigen 2052 bond.

FIG. 86C illustrates third example of cancer treatment. In FIG. 86Cmethod, part of immune system cells, for example dendritic cell 2008,may be inserted with cancer cell 2002 RNA or antigen ex vivo, so thatcell 2008 may express cancer 2008 surface antigen 2004. When dendriticcell 2008 with surface antigen 2004 is injected into PUT as in path2009, immune cell 2007 of PUT, for example T cell, may be trained ordirected as in path 2010 by the dendritic cell 2008 to recognize theexpressed cancer antigen 2004 with expressing corresponding receptor2003 on immune cell 2007. Immune cell 2007 then is able to recognizecancer cell 2002 in PUT with receptor 2003 to antigen 2004 binding 2034,and subsequently terminate cancer cell 2002.

During termination of cancer cell 2002 in FIG. 86A, FIG. 86B and FIG.86C, lysis of cancer cell 2002 occurs and genetic material, includingtumor DNA and RNA, within cancer cell 2002 is released and will finallyenter blood stream of PUT. In this invention, cell 2001 with receptor2003 of FIG. 86A, antibody 2056 and antigen 2055 of FIG. 86B, cell 2008with antigen 2004 of FIG. 86C, will be categorially referred to as“anti-tumor agent”, which may be administered externally to a PUT andcausing a target type of cancer cell 2002, if existing in PUT, toterminate and release genetic material into blood stream of PUT.

FIG. 87 illustrates first method of this invention to achievepre-symptom early stage tumor detection, which includes the sequentialsteps of: (step 1001) administer anti-tumor agent to a person-under-test(PUT) to cause termination and lysis of target tumor cells, if existingin PUT, and release of genetic material into blood stream of PUT; (step1002) collect peripheral blood from PUT; (step 1003) obtain cell-freeplasma from the collected peripheral blood; (step 1004) perform PCR onthe cell-free plasma to amplify quantity of known DNA or RNA sequencesthat identify target tumor cells, resulting in PCR sample; (step 1005)perform DNA or RNA sequencing on PCR sample and ascertain existence ofknown DNA or RNA sequence that identifies target tumor cells.

In step 1001 anti-tumor agent may be one of, or a combination of,anti-tumor agents described in FIG. 86A, FIG. 86B and FIG. 86C.Anti-tumor agent may be in sufficiently small amount that does noteliminate target tumor cells in PUT, but will cause lysis of a pluralityof target tumor cells to release genetic material to be collected instep 1003, amplified in step 1004 and detected in step 1005. Withanti-tumor agent being in sufficiently small amount, adverse effects ofFIG. 86A, FIG. 86B and FIG. 86C processes, including cytokine releasesyndrome, neurotoxicity, or off-tumor aplasia, may be limited to notcausing clinical conditions of PUT requiring medical attention. FIG. 87method implies that the DNA or RNA sequencing of step 1004 and step 1005is generic to the type of target tumor cells and is independent of PUT.

PUT of FIG. 87 may be a person without prior history of tumor, orwithout showing symptom of tumor, or without showing any physical signor results of tumor through conventional medical examination methods.PUT may be a person at risk of target tumor, for example due to geneticmutation, family history, age, environment, or occupation. PUT may be aperson who has been treated for target tumor, but needs monitor ofrecurrence of target tumor. PUT may or may not carry target tumor cells.In the case that step 1005 confirms existence of known DNA or RNAsequence that identifies target tumor cells, it may be concluded thatPUT carries target tumor, where the amount of DNA or RNA detected instep 1005 may be used to project stage and severity of tumor in PUT. Dueto the specificity of known DNA or RNA sequence that identifies targettumor, existence of such DNA or RNA may also confirm simultaneously mostprobable location of occurrence of such tumor when PUT is a pre-symptomand very early stage patient. In the case that step 1005 does not detectknown DNA or RNA sequence that identifies target tumor cells, or doesnot detect such DNA or RNA sequence at an amount that is above aconfidence threshold value, it may be concluded that PUT does not carrytarget tumor.

In practice, a time lapse may be needed after administering anti-tumoragent in step 1001, and before tumor genetic material may be releasedinto blood stream of PUT after tumor cell lysis for collection in step1002. Therefore, a scheduled waiting period, or a peripheral bloodcollection time window, may be implemented between step 1001 and step1002. Said scheduled waiting period may be between 15 minutes and 30minutes in one embodiment, between 30 minutes and 1 hour in anotherembodiment, between 1 hour and 2 hours in yet another embodiment,between 2 hours and 6 hours in yet another embodiment, between 6 hoursand 12 hours in yet another embodiment, between 12 hours and 24 hours inyet another embodiment, between 1 day and 2 days in yet anotherembodiment, between 2 days and 4 days in yet another embodiment, between4 days and 10 days in yet another embodiment, between 10 days and 15days in yet another embodiment, and between 15 days and 30 days in yetanother embodiment. Said collection time window has a start time and anend time after the time of said administering of said anti-tumor agent,where the start time and end time may respectively be 15 minutes and 30minutes in one embodiment, 30 minutes and 1 hour in another embodiment,30 minutes and 1 hour in yet another embodiment, 1 hour and 2 hours inyet another embodiment, 2 hours and 6 hours in yet another embodiment, 6hours and 12 hours in yet another embodiment, 12 hours and 24 hours inyet another embodiment, 1 day and 2 days in yet another embodiment, 2days and 4 days in yet another embodiment, 4 days and 10 days in yetanother embodiment, 10 days and 15 days in yet another embodiment, and15 days and 30 days in yet another embodiment. Additionally, amultiple-cycled repeated step 1002 to step 1005 process may be performedas shown by procedure 1009, where procedure 1009 may include a scheduledwaiting period, such that existence of target tumor cells in PUT may bemonitored and ascertained during an extended amount of time for a morecomplete anti-tumor agent action on target tumor cells. Said scheduledwaiting period in procedure 1009 may be between 6 hours and 12 hours inone embodiment, between 12 hours and 24 hours in another embodiment,between 1 day and 2 days in yet another embodiment, between 2 days and 4days in yet another embodiment, between 4 days and 10 days in yetanother embodiment, between 10 days and 15 days in yet anotherembodiment, and between 15 days and 30 days in yet another embodiment.

Advantages of FIG. 87 method are: (1) PUT may be a person carrying tumorat early stage but without tumor indication in conventional tests, thusenabling pre-symptom early stage cancer intervention; (2) byadministering anti-tumor agent targeting a specific tumor, for examplebreast cancer, pancreatic cancer, lung cancer, detection ofcorresponding tumor DNA or RNA signal also confirms type and origin ofsuch cancer, enabling fast and targeted treatment; (3) by administeringanti-tumor agent targeting a specific tumor, released DNA or RNA inblood stream may spike in a well-defined time window afterwards, whichprovides an enhanced signal-to-noise ratio (SNR) of tumor DNA or RNAdetection and may enable high sensitivity and high accuracy detectioneven when the actual amount of tumor cells in PUT is still much lowerthan being detectable by conventional tests; (4) this method may allow apanel of multiple anti-tumor agents targeting multiple types of tumorsbeing applied simultaneously to PUT to detect existence of multipletypes of target tumors at the same time, as described in FIG. 89 .Although FIG. 87 method targets pre-symptom early stage tumor, it ispossible to implement same method against dormant tumor, which may notshow fast growth, but has genetic mutation that has high risk ofmalignancy.

FIG. 88 illustrates a second method of tumor detection. FIG. 88 processis same as FIG. 87 process in every other aspect, except that: afterstep 1001, instead of collecting peripheral blood as in step 1002 ofFIG. 87 , step 1006 of FIG. 88 collects body fluid from around organ ofPUT where target tumor may occur, in which such body fluid would bewhere released DNA or RNA by lysed target tumor cells may be found, forexample urine for bladder cancer, or prostate secretion for prostatecancer; then step 1007 replaces step 1003 of FIG. 87 , and obtainscell-free fluid from the collected body fluid of step 1006; and thenstep 108 replaces step 1004 of FIG. 87 , and performs PCR on cell-freefluid to amplify quantity of known DNA or RNA sequences that identifytarget tumor cells, resulting in PCR sample. PCR sample of step 1008then undergoes same step 1005 as in FIG. 87 .

FIG. 89 illustrates a third method of tumor detection. FIG. 89 processis same as FIG. 87 process, but expanding tumor detection from onetarget tumor to multiple types of tumor. FIG. 89 process includes thesequential steps of: (step 3001) administer anti-tumor agents to PUT tocause lysis of a plurality types of target tumor cells to releasegenetic material into blood stream of PUT; (step 1002) collectperipheral blood from PUT; (step 1003) obtain cell-free plasma from thecollected peripheral blood; (step 3004) perform PCR on the cell-freeplasma to amplify quantity of known DNA or RNA sequences that identifyeach type of the plurality types of target tumor cells, resulting in PCRsample; (step 3005) perform DNA or RNA sequencing on PCR sample andascertain existence of known DNA or RNA sequence that identifies eachtype of the plurality types of target tumor cells. In FIG. 89 ,ascertaining existence of any of the multiple types of tumors may beperformed at same time in PUT. Anti-tumor agents in step 3001 may be oneof anti-tumor agents described in FIG. 86A, FIG. 86B and FIG. 86C, whichlyses multiple types of tumors simultaneously. Anti-tumor agents in step3001 may be a combination of more than one anti-tumor agents describedin FIG. 86A, FIG. 86B and FIG. 86C, where each different anti-tumoragent lyses one type, or a sub-set of types, of the plurality types oftumors. Steps 1002 and 1003 of FIG. 89 may be replaced by steps 1006 and1007 of FIG. 88 to collect body fluids, where step 3004 maycorrespondingly be updated with performing PCR on cell-free fluid fromstep 1007.

FIG. 90 , FIG. 91 , and FIG. 92 illustrate embodiments of process flowsto utilize MAG and UFL devices to obtain cell-free plasma fromperipheral blood. For simplicity of description, terms UFL and MAG areused in these figures for explanation. However, UFL may be any of UFL600, 620, 630, 640 of FIG. 40A, FIG. 41A, FIG. 42A, FIG. 43 , while MAGmay be any of MAG 121, 122, 123, 124, 124,125, 126, 127, 128, 129 withcorresponding channel types as described in prior figures withoutlimitation and without sacrifice of performance.

FIG. 90 illustrates embodiment of first process flow to obtain cell-freeplasma of step 1003 from collected peripheral blood in step 1002. Aftercollection of peripheral blood in step 1002, peripheral blood may becentrifuged as in step 5001, and result of centrifuge may containcell-free plasma that may directly achieve step 1003 as indicated bypath 5005. Alternatively, after centrifuge step 5001, blood plasma maybe depleted of certain blood cells, for example red blood cells, but maynot have enough purity for later stage PCR process. Plasma from step5001 may then be sent along path 5008 to undergo a UFL separation instep 5002, where UFL large entity output contains any remaining cells inplasma, while UFL small entity output contains DNA or RNA and may bemore concentrated than plasma from step 5001. In yet another alternativepath, peripheral blood from step 1002 may skip step 5001 and directlyinput into UFL separation step 5002 as shown by path 5007, where the UFLseparates cells through large entity output while maintaining DNA andRNA in small entity output. Small entity output from UFL separation ofstep 5002 may then be used towards step 1003 as cell-free plasmaaccording to path 5009. Further alternatively, magnetic labelshybridized with antibodies or ligands may be added to the plasma fromUFL small entity output of step 5002 to bind to DNA or RNA within theplasma as in step 5003. MAG device may be used to separate the DNA andRNA bound with the magnetic labels in step 5004, and the resultingpositive MAG sample containing DNA and RNA may then be regarded as thecell-free plasma of step 1003.

FIG. 91 illustrates embodiment of second process flow to obtaincell-free plasma of step 1003 from collected peripheral blood in step1002. After collection of peripheral blood in step 1002, peripheralblood may be centrifuged as in step 5001, and resulting blood plasma maybe depleted of certain blood cells, for example red blood cells. Then instep 5003, magnetic labels hybridized with antibodies or ligands may beadded to the plasma from step 5001 to bind to DNA or RNA within theplasma. Alternatively, peripheral blood from step 1002 may be useddirectly in step 5003 without step 5001 centrifuge as shown by path5015, and magnetic labels bind to the DNA or RNA in peripheral blood instep 5003. After step 5003, MAG device may be used to separate the DNAand RNA bound with the magnetic labels in step 5004, and the resultingpositive MAG sample containing DNA and RNA may be regarded as thecell-free plasma of step 1003 as indicated by path 5013. Furtheralternatively, positive MAG sample of step 5004 may undergo a UFLseparation in step 5002, where UFL large entity output contains anyremaining cells in plasma, while UFL small entity output contains DNA orRNA that are bound with magnetic labels. Small entity output from UFLseparation of step 5002 may then be used towards step 1003 as cell-freeplasma according to path 5017.

FIG. 92 illustrates embodiment of third process flow to obtain cell-freeplasma of step 1003 from collected peripheral blood in step 1002. Aftercollection of peripheral blood in step 1002, peripheral blood may becentrifuged as in step 5001, and resulting blood plasma may be depletedof certain blood cells, for example red blood cells. Then in step 5023,magnetic labels hybridized with antibodies or ligands may be added tothe plasma from step 5001 to bind to cells within the plasma.Alternatively, peripheral blood from step 1002 may be used directly instep 5003 without step 5001 centrifuge as shown by path 5015, andmagnetic labels bind to the cells in peripheral blood in step 5023.After step 5023, MAG device may be used to separate the cells bound withthe magnetic labels in step 5024 from the plasma that contains DNA orRNA, and the resulting negative MAG sample containing DNA and RNA may beregarded as the cell-free plasma of step 1003 as indicated by path 5013.Alternatively, negative MAG sample of step 5024 may undergo a UFLseparation in step 5002, where UFL large entity output contains anyremaining cells in plasma from step 5023, while UFL small entity outputcontains DNA or RNA. Small entity output from UFL separation of step5002 may then be used towards step 1003 as cell-free plasma according topath 5017.

FIG. 93 illustrates a method to extend method of FIG. 89 for anti-agingpurpose. For conditions relating to aging, for example Alzheimer'sDisease, dementia, osteoporosis, and arthritis, human growth hormone orother anti-aging agents may be used to help cell growth to delay oralleviate symptom occurrence of the conditions. For aging in general,human growth hormone or other anti-aging agents may help to improveoverall body conditions due to tissue or cell replenishment. However, inusing such anti-aging agents, a possible limitation is increased risk oftumor occurrence. Due to the nature of tumor cells being fast growth andevasive to cell death by immune system, administering human growthhormone or anti-aging agent in presence of any tumor, especiallypre-symptom tumor or dormant tumor, such hormone or agent may incurgrowth of these tumor cells. Thus, for low risk implementation of humangrowth hormone or anti-aging agent, a tumor free condition of PUT isdesired. FIG. 93 initial flow steps of 3001, 1002, 1003, 3004 and 3005are identical to FIG. 89 , where existence of any of the multiple typesof target tumors in PUT may be ascertained in step 3005. In FIG. 93 ,after step 3005, in the case that at least one type of tumor isconfirmed to exist in PUT as in judgement 4001, corresponding tumortreatment may be performed in step 4002. After step 4002, another cycleof process from step 3001 to step 3005 may be performed to ascertaintumor absence after treatment of step 4002. In the case that step 3005finds no tumor existence as in judgement 4003, growth hormone oranti-aging agent may be administered to the PUT as in step 4004. Afterstep 4004, another cycle of step 3001 through step 3005 may be performedto ascertain no tumor was promoted by the step 4004. Flow from step 3001to step 4004 may be repeated as many cycles as needed to achieve agingrelated condition treatment goal.

While the current invention has been shown and described with referenceto certain embodiments, it is to be understood that those skilled in theart will no doubt devise certain alterations and modifications theretowhich nevertheless include the true spirit and scope of the currentinvention. Thus the scope of the invention should be determined by theappended claims and their legal equivalents, rather than by examplesgiven.

What is claimed is:
 1. A device for separating biological entities froma sample fluid, the device comprising: a substrate including: a linearchannel having a wide section with a first constant width, a narrowsection with a second constant width, and a tapered transition sectionconnecting a downstream end of said wide section to an upstream end ofsaid narrow section; a first inlet connected to an upstream end of saidwide section for introducing a buffer fluid into said linear channel; afirst outlet connected to a downstream end of said narrow section; asecond inlet for introducing said sample fluid into said wide section; afirst pair of side channels connecting said second inlet to saidupstream end of said wide section; a second outlet; and a second pair ofside channels connecting said second outlet to said downstream end ofsaid narrow section; and a means for generating ultrasound in a form ofa standing wave in said sample and buffer fluids in said wide and narrowsections to separate said biological entities by size, wherein saidfirst constant width is a first integer multiple of one-half wavelengthof said standing wave, and said second constant width is a secondinteger multiple of one-half wavelength of said standing wave.
 2. Thedevice according to claim 1, wherein said linear channel, said firstinlet, said second inlet, said first outlet, said second outlet, saidfirst pair of side channels, and said second pair of side channels arerecessed into said substrate from a first surface of said substrate. 3.The device according to claim 2, further comprising a cover attached tosaid first surface to form a first wall of said linear channel.
 4. Thedevice according to claim 3, wherein said means for generatingultrasound comprises a piezoelectric transducer attached to a surface ofsaid cover opposite said first wall.
 5. The device according to claim 3,wherein said cover includes through-holes aligned with said first inlet,said second inlet, said first outlet, and said second outlet,respectively.
 6. The device according to claim 1, wherein said means forgenerating ultrasound comprises a piezoelectric transducer attached tosaid substrate.
 7. The device according to claim 1, wherein saidbiological entities exiting said first outlet are larger than saidbiological entities exiting said second outlet.
 8. The device accordingto claim 1, wherein said buffer fluid forms a buffer flow at a center ofsaid linear channel and said sample fluid forms sample flows along twoside walls of said linear channel, respectively.
 9. The device accordingto claim 1, wherein a multiplier of said second integer multiple is one.10. The device according to claim 1, wherein said biological entitiesare bound with fluorescent labels.
 11. The device according to claim 1,wherein said biological entities are bound with magnetic labels.
 12. Thedevice according to claim 1, wherein said biological entities are anyone of: cells, bacteria, particles, DNA, RNA, and molecules.
 13. Thedevice according to claim 1, wherein said substrate further includes athird pair of side channels connecting said second outlet to saiddownstream end of said wide section.
 14. The device according to claim13, wherein a group of said biological entities enter said third pair ofside channels from said wide section, and wherein said group of saidbiological entities have a smaller physical size from among saidbiological entities.
 15. The device according to claim 1, wherein saidsubstrate further includes a third pair of side channels connecting saidsecond pair of side channels to said downstream end of said widesection.
 16. A device for separating biological entities from a samplefluid, the device comprising: a substrate including: a linear channelhaving an input end, an output end, a plurality of channel sections withdifferent constant widths between said input and output ends, and one ormore tapered transition sections with each tapered transition sectioninterposed between two channel sections of said plurality of channelsections; a first inlet connected to said linear channel at said inputend for introducing a buffer fluid into said linear channel; a secondinlet for introducing said sample fluid into said linear channel; afirst pair of side channels connecting said second inlet to said linearchannel at said input end; a first outlet connected to said linearchannel at said output end; a second outlet; and a second pair of sidechannels connecting said second outlet to said linear channel at saidoutput end; and a means for generating ultrasound in a form of astanding wave in said sample and buffer fluids in said plurality ofchannel sections to separate said biological entities by size, whereineach of said plurality of channel sections has a constant width that isan integer multiple of one-half wavelength of said standing wave, andsaid plurality of channel sections become progressively narrower fromsaid input end to said output end.
 17. The device according to claim 16,wherein said substrate further includes a third pair of side channelsconnecting said second pair of side channels to one of said plurality ofchannel sections at a downstream location adjacent to one of said one ormore tapered transition sections.
 18. The device according to claim 17,wherein said substrate further includes a fourth pair of side channelsconnecting said third pair of side channels to another one of saidplurality of channel sections at another downstream location adjacent toanother one of said one or more tapered transition sections.
 19. Thedevice according to claim 16, wherein said biological entities exitingsaid first outlet are larger than said biological entities exiting saidsecond outlet.
 20. A method for separating biological entities from asample fluid using a microfluidic device comprising the steps of:passing said sample fluid and a buffer fluid into a linear channelhaving an input end, an output end, a plurality of channel sections withdifferent constant widths between said input and output ends, and one ormore tapered transition sections with each tapered transition sectioninterposed between two channel sections of said plurality of channelsections, wherein said buffer fluid flows along a center of said linearchannel and said sample fluid flows along two side walls of said linearchannel; generating ultrasound in a form of a standing wave in saidsample and buffer fluids in said plurality of channel sections; passinga first group of said biological entities through a pair of sidechannels connected to one of said plurality of channel sections at adownstream end adjacent to one of said one or more tapered transitionsections; and passing a second group of said biological entities to asecond outlet connected to said output end, wherein said first group ofsaid biological entities are smaller in physical size than said secondgroup of said biological entities, and wherein said plurality of channelsections become progressively narrower from said input end to saidoutput end.